Multiple signaling pathways induced by hexavalent, monospecific and bispecific antibodies for enhanced toxicity to B-cell lymphomas and other diseases

ABSTRACT

Disclosed herein are compositions and methods of use comprising hexavalent DNL complexes. Preferably, the complexes comprise anti-CD20 and/or anti-CD22 antibodies or fragments thereof. More preferably, the anti-CD20 antibody is veltuzumab and the anti-CD22 antibody is epratuzumab. Administration of the subject hexavalent DNL complexes induces apoptosis and cell death of target cells in diseases such as B-cell lymphomas or leukemias, autoimmune disease or immune dysfunction disease. In most preferred embodiments, the DNL complexes increase levels of phosphorylated p38 and PTEN, decrease levels of phosphorylated Lyn, Akt, ERK, IKKα/β and IκBα, increase expression of RKIP and Bax and decrease expression of Mcl-1, Bcl-xL, Bcl-2, and phospho-BAD in target cells. The subject DNL complexes show EC 50  values for inhibiting tumor cell growth in the low nanomolar or even sub-nanomolar concentration range.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Application Ser. No. 61/324,111, filed Apr. 14,2010. This application is a continuation-in-part of U.S. patentapplication Ser. Nos. 13/036,820, filed Feb. 28, 2011; 13/021,302, filedFeb. 4, 2011, (which was a divisional of U.S. Pat. No. 7,906,121); whichwas a divisional of U.S. Pat. No. 7,534,866); 13/012,977, filed Jan. 25,2011, (which was a divisional of U.S. Pat. No. 7,906,118); 13/010,993,filed Jan. 21, 2011, (which was a divisional of U.S. Pat. No.7,901,680); 13/004,349, filed Jan. 11, 2011; 12/968,936, filed Dec. 15,2010, (which was a divisional of U.S. Pat. No. 7,871,622; which was adivisional of U.S. Pat. No. 7,521,056); 12/964,021, filed Dec. 9, 2010;12/949,536, filed Nov. 18, 2010, (which was a divisional of U.S. Pat.No. 7,858,070; which was a divisional of U.S. Pat. No. 7,527,787);12/915,515, filed Oct. 29, 2010; 12/871,345, filed Aug. 30, 2010;12/869,823, filed Aug. 27, 2010; 12/754,740, filed Apr. 6, 2010;12/752,649, filed Apr. 1, 2010; 12/731,781, filed Mar. 25, 2010;12/644,146, filed Dec. 22, 2009, (which was a divisional of U.S. Pat.No. 7,666,400); and 12/468,589, filed May 19, 2009, (which was adivisional of U.S. Pat. No. 7,550,143). Those applications claimed thebenefit under 35 U.S.C. 119(e) of provisional U.S. Patent Applications61/414,592, filed Nov. 17, 2010; 61/378,059, filed Aug. 30, 2010;61/374,449, filed Aug. 17, 2010; 61/323,960, filed Apr. 14, 2010;61/323,001, filed Apr. 12, 2010; 61/316,996, filed Mar. 24, 2010;61/302,682, filed Feb. 9, 2010; 61/293,846, filed Jan. 11, 2010;61/267,877, filed Dec. 9, 2009; 61/266,305, filed Dec. 3, 2009;61/258,729, filed Nov. 6, 2009; 61/258,369, filed Nov. 5, 2009;61/238,424, filed Aug. 31, 2009; 61/238,473, filed Aug. 31, 2009;61/168,668, filed Apr. 13, 2009; 61/168,657, filed Apr. 13, 2009;61/168,290, filed Apr. 10, 2009; 61/163,666, filed Mar. 26, 2009;61/119,542, filed Dec. 3, 2008; 61/104,916, filed Oct. 13, 2008;61/090,487, filed Aug. 20, 2008; 61/043,932, filed Apr. 10, 2008;60/864,530, filed Nov. 6, 2006; 60/782,332, filed Mar. 14, 2006;60/751,196, filed Dec. 16, 2005; 60/728,292, filed Oct. 19, 2005;60/668,603, filed Apr. 6, 2005.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 27, 2011, isnamed IBC127US.txt and is 54,202 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compositions and methods of use ofhexavalent dock-and-lock (DNL) constructs, comprising antibodies and/orantigen-binding antibody fragments. Preferably, the antibodies orfragments thereof bind to CD20 and/or CD22. The compositions and methodsare of use for therapy of autoimmune disease, immune dysfunctiondisease, B-cell lymphomas, B-cell leukemias and other conditions inwhich disease-associated cells express the target antigens. In preferredembodiments, the compositions and methods exhibit enhanced toxicity toCD20 and/or CD22 expressing target cells, by inducing multiple signalingpathways in the target cell. Such pathways may include, but are notlimited to, increased levels of phosphorylated p38, increased levels ofPTEN (phosphatase and tensin homolog deleted on chromosome 10),increased apoptosis, decreased levels of phosphorylated Lyn, Akt, ERK,IKKα/β and IκBα, increased expression of RKIP and Bax and decreasedexpression of Mcl-1, Bcl-xL, Bcl-2, and phospho-BAD. In most preferredembodiments, the compositions and methods are cytotoxic to diseaseassociated cells that express CD20 and/or CD22, with EC₅₀ values in theabsence of cross-linking antibodies in the low nanomolar orsub-nanomolar range.

BACKGROUND

To address the clinical concerns of immunogenicity and suboptimalpharmacokinetics, cancer therapy with monoclonal antibodies has evolvedfrom murine to chimeric, humanized, and fully human constructs. Parallelto these improvements have been continuing efforts to develop moreeffective forms of antibodies, which to date include different antibodyisotypes, single-chain antibody fragments with monomeric or multimericbinding moieties, specific mutations in the Fc region to modulateeffector function or circulating half-life, and bispecific antibodies ofnumerous designs that vary in valency, structure, and constituents(Chames et al., Br J Pharmacol 2009, 157:220-233).

Because signaling pathway redundancies can result in lack of response toa single antibody, diverse strategies to use combination therapy withantibodies that bind to different epitopes or different antigens on thesame target cell have been proposed. Combinations such as anti-CD20 andanti-CD22 (Stein et al., Clin Cancer Res 2004, 10:2868-2878), anti-CD20and anti-HLA-DR (Tobin et al, Leuk Lymphoma 2007, 48:944-956), anti-CD20and anti-TRAIL-R1 (Maddipatla et al., Clin Cancer Res 2007,13:4556-4564), anti-IGF-1R and anti-EGFR (Goetsche et al., Int J Cancer2005, 113:316-328), anti-IGF-1R and anti-VEGF (Shang et al., Mol CancerTher 2008, 7:2599-2608), or trastuzumab and pertuzumab that targetdifferent regions of human EGFR2 (Nahta et al., Cancer Res 2004,64:2343-2346) have been evaluated preclinically, showing enhanced orsynergistic antitumor activity in vitro and in vivo.

The first clinical evidence of an apparent advantage of combining twoantibodies against different cancer cell antigens involved theadministration of rituximab (chimeric anti-CD20) and epratuzumab(humanized anti-CD22 antibody) in patients with non-Hodgkin lymphoma(NHL). The combination was found to enhance anti-lymphoma efficacywithout a commensurate increase in toxicity, based on 3 independentclinical trials (Leonard et al., J Clin Oncol 2005, 23:5044-5051).

Given the number of antibodies approved for cancer therapy, the numberof such potential combinations is not large. However, where suchcombinations show improved efficacy, there is concern over the combinedcost of individually expensive antibody therapies, in addition to theinconvenience and time of conducting separate infusions. As analternative, attempts to develop bispecific antibodies that cansimultaneously bind two target antigens have resulted in a multitude ofapproaches (Chames & Baty, Curr Opin Drug Discov Devel 2009,12:276-283).

Earlier methods used for the production of bispecific antibodies madeuse of chemical cross-linking of IgG or Fab′ (Perez et al., Nature 1985,316:354-356; Glennie et al., J Immunol 1987, 139:2367-2375) or quadromasobtained by fusing two hybridomas together (Staerz & Bevan, Proc NatlAcad Sci USA 1986, 83:1453-1457). Subsequent strategies focused ongenerating recombinant bispecific antibodies composed of tandem scFvs ordiabodies (Kriangkum et al., Biomol Eng 2001, 18:31-40). One format ofsuch Fc-lacking constructs, referred to as BiTe, is currently beingtested clinically (Baeuerle & Reinhardt, Cancer Res 2009, 69:4941-4944).Because the presence of an Fc region and its effector functions showsimproved in vivo properties for many therapeutic applications, a varietyof Fc-containing bispecific antibody designs have also have beensuggested (Coloma & Morrison, Nat Biotechnol 1997, 15:159-163; Shen etal., J Biol Chem 2006, 281:10706-10714; Asano et al., J Biol Chem 2007,282:27659-27665; Wu et al., Nat Biotechnol 2007, 25:1290-1297).

We have developed a novel approach for constructing multivalentantibodies using the dock-and-lock (DNL) method (Rossi et al., Proc NatlAcad Sci USA 2006, 103:6841-6846), which enables site-specificself-assembly of two modular components only with each other. The DNLmethod results in a covalent structure of defined composition withretained bioactivity (Chang et al., Clin Cancer Res 2007,13:5586s-5591s). Since the co-administration of anti-CD20 and anti-CD22antibodies showed improved anti-lymphoma efficacy without increasedtoxicity in patients (Leonard et al., J Clin Oncol 2005, 23:5044-5051;Leonard & Goldenberg, Oncogene 2007, 26:3704-3713), and enhancedactivity in a lymphoma xenograft model (Stein, et al., Clin Cancer Res2004, 10:2868-2878), the studies reported in the present applicationutilized the DNL technique to develop hexavalent, monospecific anti-CD20or bispecific anti-CD20/22 constructs with improved pharmacokineticproperties, increased efficacy and novel mechanisms of action forkilling B-cell lymphoma, leukemia or other target cells.

SUMMARY

The present invention concerns improved compositions and methods of useof multivalent, monospecific or bispecific antibodies for therapy ofB-cell lymphoma, leukemia and other conditions in whichdisease-associated cells express target antigens. Preferably, theantibodies are monospecific for CD20 or bispecific for CD20/CD22. Inmore preferred embodiments, the constructs are hexavalent constructsmade by the dock-and-lock (DNL) technique (see, e.g., U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,901,680; 7,906,118 and 7,906,121, the Examples section ofeach of which is incorporated herein by reference.) The DNL techniquetakes advantage of the specific, high-affinity binding interactionbetween a dimerization and docking domain (DDD) sequence from theregulatory subunit of human cAMP-dependent protein kinase (PKA), such ashuman PKA RIα, RIβ, RIIα or RIIβ, and an anchor domain (AD) sequencefrom any of a variety of AKAP proteins. The DDD and AD peptides may beattached to any protein, peptide or other molecule. Because the DDDsequences spontaneously dimerize and bind to the AD sequence, the DNLtechnique allows the formation of complexes between any selectedmolecules that may be attached to DDD or AD sequences. Although thestandard DNL complex comprises a trimer with two DDD-linked moleculesattached to one AD-linked molecule, variations in complex structureallow the formation of dimers, trimers, tetramers, pentamers, hexamersand other multimers. In some embodiments, the DNL complex may comprisetwo or more antibodies, antibody fragments or fusion proteins which bindto the same antigenic determinant or to two or more different antigens.The DNL complex may also comprise one or more other effectors, such as acytokine or PEG moiety.

In preferred embodiments, the hexavalent DNL constructs comprise an IgGmolecule covalently attached to two copies of an AD moiety, which bindsto four Fab fragments, each covalently attached to a DDD moiety. Thehexavalent DNL construct therefore comprises six Fab moieties attachedto an Fc moiety. By formation of disulfide bonds between the AD and DDDsubunits, the entire DNL complex is highly stable under in vivoconditions and each Fab moiety retains the binding specificity andaffinity of the parent antibody. In alternative embodiments, thehexavalent DNL construct may comprise 6 anti-CD20 Fabs; 4 anti-CD20 and2 anti-CD22 Fabs; or 2 anti-CD20 and 4 anti-CD22 Fabs, attached to an Fcmoiety. In most preferred embodiments, the hexavalent constructs maycomprise the anti-CD22 IgG antibody epratuzumab attached to four Fabsubunits of the anti-CD20 antibody veltuzumab (designated 22-20); theveltuzumab IgG attached to four Fab subunits of epratuzumab (designated20-22); or the veltuzumab IgG attached to four Fab subunits ofveltuzumab (designated 20-20) (Rossi et al., Blood 2009, 113:6161-6171;Rossi et al., Cancer Res 2008, 68:8384-8392). Previous studies haveshown that 22-20, 20-22, and 20-20 have distinct properties comparedwith their parental counterparts, including enhanced anti-lymphomaactivity in vitro and comparable efficacy in vivo, despite showingshorter circulating half-lives (Rossi et al., Blood 2009, 113:6161-6171;Rossi et al., Cancer Res 2008, 68:8384-8392).

Many examples of anti-CD20 antibodies are known in the art and any suchknown antibody or fragment thereof may be utilized. In a preferredembodiment, the anti-CD20 antibody is an hA20 antibody (also known asveltuzumab) that comprises the light chain complementarity-determiningregion (CDR) sequences CDR1 (RASSSVSYIH; SEQ ID NO:1), CDR2 (ATSNLAS;SEQ ID NO:2), and CDR3 (QQWTSNPPT; SEQ ID NO:3) and the heavy chainvariable region CDR sequences CDR1 (SYNMH; SEQ ID NO:4), CDR2(AIYPGNGDTSYNQKFKG; SEQ ID NO:5), and CDR3 (STYYGGDWYFDV; SEQ ID NO:6).

A humanized anti-CD20 antibody suitable for use is disclosed in U.S.Pat. No. 7,435,803, incorporated herein by reference from Col. 36, line4 through Col. 46, line 52 and FIGS. 1, 2, 4, 5 and 7. However, inalternative embodiments, other known and/or commercially availableanti-CD20 antibodies may be utilized, such as rituximab; ofatumumab;ibritumomab; tositumomab; ocrelizumab; GA101; BCX-301; DXL 625; L26,B-Ly1, MEM-97, LT20, 2H7, AT80, B-H20 (ABCAM®, Cambridge, Mass.); HI20a,HI47, 13.6E12 (ABBIOTEC®, San Diego, Calif.); 4f11, 5c11, 7d1 (ABDSEROTEC®, Raleigh, N.C.) and any other anti-CD20 antibody known in theart.

The anti-CD20 antibody may be selected such that it competes with orblocks binding to CD20 of an hA20 antibody comprising the light chaincomplementarity-determining region (CDR) sequences CDR1 (RASSSVSYIH; SEQID NO:1), CDR2 (ATSNLAS; SEQ ID NO:2), and CDR3 (QQWTSNPPT; SEQ ID NO:3)and the heavy chain variable region CDR sequences CDR1 (SYNMH; SEQ IDNO:4), CDR2 (AIYPGNGDTSYNQKFKG; SEQ ID NO:5), and CDR3 (STYYGGDWYFDV;SEQ ID NO:6). Alternatively, the anti-CD20 antibody may bind to the sameepitope of CD20 as a hA20 antibody.

Many examples of anti-CD22 antibodies are also known in the art and anysuch known antibody or fragment thereof may be utilized. In a preferredembodiment, the anti-CD22 antibody is an hLL2 antibody (also known asepratuzumab) that comprises the light chain CDR sequences CDR1(KSSQSVLYSANHKYLA, SEQ ID NO:7), CDR2 (WASTRES, SEQ ID NO:8), and CDR3(HQYLSSWTF, SEQ ID NO:9) and the heavy chain CDR sequences CDR1 (SYWLH,SEQ ID NO:10), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:11), and CDR3(RDITTFY, SEQ ID NO:12). A humanized LL2 anti-CD22 antibody suitable foruse is disclosed in U.S. Pat. No. 6,187,287, incorporated herein byreference from Col. 11, line 40 through Col. 20, line 38 and FIGS. 1, 4and 5. However, in alternative embodiments, other known and/orcommercially available anti-CD22 antibodies may be utilized, such as1F5; HIB22 (ABBIOTEC®, San Diego, Calif.); FPC1, LT22, MEM-1, RFB4(ABCAM®, Cambridge, Mass.); bu59, fpc1, mc64-12 (ABD SEROTEC®, Raleigh,N.C.); IS7 (ABNOVA®, Taipei City, Taiwan) and any other anti-CD22antibody known in the art.

The anti-CD22 antibody may be selected such that it competes with orblocks binding to CD22 of an LL2 antibody comprising the light chain CDRsequences CDR1 (KSSQSVLYSANHKYLA, SEQ ID NO:7), CDR2 (WASTRES, SEQ IDNO:8), and CDR3 (HQYLSSWTF, SEQ ID NO:9) and the heavy chain CDRsequences CDR1 (SYWLH, SEQ ID NO:10), CDR2 (YINPRNDYTEYNQNFKD, SEQ IDNO:11), and CDR3 (RDITTFY, SEQ ID NO:12). Alternatively, the anti-CD22antibody may bind to the same epitope of CD22 as an LL2 antibody.

The anti-CD20 and/or anti-CD22 antibodies or fragments thereof may beused as naked antibodies, alone or in combination with one or moretherapeutic agents. Alternatively, the antibodies or fragments may beutilized as immunoconjugates, attached to one or more therapeuticagents. (For methods of making immunoconjugates, see, e.g., U.S. Pat.Nos. 4,699,784; 4,824,659; 5,525,338; 5,677,427; 5,697,902; 5,716,595;6,071,490; 6,187,284; 6,306,393; 6,548,275; 6,653,104; 6,962,702;7,033,572; 7,147,856; and 7,259,240, the Examples section of eachincorporated herein by reference.) Therapeutic agents may be selectedfrom the group consisting of a radionuclide, a cytotoxin, achemotherapeutic agent, a drug, a pro-drug, a toxin, an enzyme, animmunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, acytokine, a hormone, an oligonucleotide molecule (e.g., an antisensemolecule or a gene) or a second antibody or fragment thereof.

The therapeutic agent may be selected from the group consisting ofaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,cisplatinum, irinotecan (CPT-11), SN-38, carboplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes,paclitaxel, testosterone propionate, thalidomide, thioguanine, thiotepa,teniposide, topotecan, uracil mustard, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, ranpirnase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

The therapeutic agent may comprise a radionuclide selected from thegroup consisting of ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt,¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ¹¹C, ^(121m)Te, ^(122m)Te, ¹²⁵I,^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹³N, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm,¹⁵O, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au,¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po,²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²²⁵Fm, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr,⁵⁷Cr, ⁵⁷Co, 58Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br,^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo and ^(99m)Tc.

The therapeutic agent may be an enzyme selected from the groupconsisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

An immunomodulator of use may be selected from the group consisting of acytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and combinations thereof. Exemplaryimmunomodulators may include IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,IL-18, IL-21, interferon-α, interferon-β, interferon-γ, G-CSF, GM-CSF,and mixtures thereof.

Exemplary anti-angiogenic agents may include angiostatin, endostatin,basculostatin, canstatin, maspin, anti-VEGF binding molecules,anti-placental growth factor binding molecules, or anti-vascular growthfactor binding molecules.

In certain embodiments, the antibody or fragment may comprise one ormore chelating moieties, such as NOTA, DOTA, DTPA, TETA, Tscg-Cys, orTsca-Cys. In certain embodiments, the chelating moiety may form acomplex with a therapeutic or diagnostic cation, such as Group II, GroupIII, Group IV, Group V, transition, lanthanide or actinide metalcations, Tc, Re, Bi, Cu, As, Ag, Au, At, or Pb.

In some embodiments, the antibody or fragment thereof may be a human,chimeric, or humanized antibody or fragment thereof. A humanizedantibody or fragment thereof may comprise thecomplementarity-determining regions (CDRs) of a murine antibody and theconstant and framework (FR) region sequences of a human antibody, whichmay be substituted with at least one amino acid from corresponding FRsof a murine antibody. A chimeric antibody or fragment thereof mayinclude the light and heavy chain variable regions of a murine antibody,attached to human antibody constant regions. The antibody or fragmentthereof may include human constant regions of IgG1, IgG2a, IgG3, orIgG4. Exemplary known antibodies of use include, but are not limited to,hR1 (anti-IGF-1R), hPAM4 (anti-mucin), hA20 (anti-CD20), hA19(anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22),hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15(anti-CEACAM6), 29H2 (anti-CEACAM1, ABCAM®), hRS7 (anti-EGP-1) and hMN-3(anti-CEACAM6).

Although in preferred embodiments that antibodies or fragments thereofincorporated into the hexavalent constructs bind to CD20 and/or CD22, inalternative embodiments antibodies or fragments may bind to one or moretarget antigens selected from the group consisting of carbonic anhydraseIX, alpha-fetoprotein, α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CXCR4,colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM1, CEACAM6, c-met,DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folatereceptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-α, IFN-β,IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-25, insulin-like growth factor-1 (IGF-1),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3,mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1/2,MUM-3, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody,placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA,PRAME, PSMA, P1GF, IGF, IGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE,5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors,TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complementfactors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6,Kras, cMET, an oncogene marker and an oncogene product (see, e.g., Sensiet al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol2007, 178:1975-79; Novellino et al. Cancer Immunol Immunother 2005,54:187-207). Reports on tumor associated antigens include Mizukami etal., (2005, Nature Med. 11:992-97); Hatfield et al., (2005, Curr. CancerDrug Targets 5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol.23:3536-44); and Ren et al. (2005, Ann. Surg. 242:55-63).

Also disclosed is a method for treating and/or diagnosing a disease ordisorder that includes administering to a patient a therapeutic and/ordiagnostic composition that includes any of the aforementionedantibodies or fragments thereof. Typically, the composition isadministered to the patient intravenously, intramuscularly orsubcutaneously at a dose of 20-5000 mg. In preferred embodiments, thedisease or disorder is a B-cell lymphoma or leukemia, an immunedysregulation disease, an autoimmune disease, organ-graft rejection orgraft-versus-host disease. More preferably, the disease is a B-celllymphoma or leukemia. Exemplary malignancies that may be treated usingthe claimed methods and compositions include, but are not limited to,indolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, acute lymphocytic leukemia, chronic lymphocytic leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma,diffuse large B-cell lymphoma, follicular lymphoma, marginal zonelymphoma, Burkitt's lymphoma and multiple myeloma

Exemplary autoimmune diseases include acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, or fibrosing alveolitis.

In particularly preferred embodiments, administration DNL complexescomprising anti-CD20 and/or anti-CD22 antibodies or fragments thereofcan deplete disease-associated cells by mechanisms including, but notlimited to, increasing levels of phosphorylated p38, increasing levelsof PTEN, increasing apoptosis, decreasing levels of phosphorylated Lyn,Akt, ERK, IKKα/β and IκBα, increasing expression of RKIP and Bax anddecreasing expression of Mcl-1, Bcl-xL, Bcl-2, and phospho-BAD.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are provided to illustrate exemplary, butnon-limiting, preferred embodiments of the invention. The followingterms were used interchangeably in designating hexavalent DNLconstructs.

-   -   20-20: Also referred to as Hex-hA20, comprising an anti-CD20        hA20 IgG attached to four anti-CD20 hA20 Fab moieties.    -   20-22: Also referred to as DNL2, comprising an anti-CD20 hA20        IgG attached to four anti-CD22 hLL2 Fab moieties.    -   22-20: Also referred to as DNL1, comprising an anti-CD22 hLL2        IgG attached to four anti-CD20 hA20 Fab moieties.

FIG. 1. Competitive ELISA experiments to compare the relative hA20/hLL2binding avidities of DNL1, DNL2, Hex-hA20 and Hex-hLL2 with the parentalIgGs. Microtiter plates were coated with hA20 or hLL2 IgG at 5 μg/ml.Dilution series of the HIDS were mixed with anti-Ids specific to hA20 orhLL2 IgG, which was maintained at a constant concentration (2 nM). Thelevel of binding of the anti-Ids to the coated wells was detected usingperoxidase-conjugated-Goat anti-Rat IgG and OPD substrate solution. Theresults are plotted as % inhibition (of anti-Id binding to coated wells)vs. concentration of HIDS. EC₅₀ (the effective concentration resultingin 50% inhibition) values were derived using Prism software. The HIDSwere used to compete for binding to (A) WI2 (hA20 Rat anti-Id) inhA20-coated wells or (B) WN (hLL2 Rat anti-Id) in hLL2-coated wells.

FIG. 2. Dose-response experiment for treatment of Daudi cells withvarious HIDS. Cells were plated in 96-well plates at 5,000 cells/well inRPMI 1640 media. Five-fold serial dilutions were performed in triplicatefrom concentrations of 2×10⁻⁸ down to 6.4×10⁻¹²M. The plates wereincubated for four days, after which MTS reagent was added and theincubation was continued for an additional four hours before reading theplates at 490 nm. The results are given as percent of the OD₄₉₀ foruntreated wells vs. the log of the molar concentration of HIDS. EC₄₀(the effective concentration resulting in 40% growth inhibition) valueswere measured for each dose-response curve.

FIG. 3. In vivo therapy of mice bearing human Burkitt Lymphoma (Daudi)treated with DNL2 or Hex-hA20. Mice (4/group) were inoculated i.v. with1.5×10⁷ Daudi cells (day 0). On days 1, 4 and 7, mice were administeredeither 4 μg or 20 μg of DNL2 or Hex-hA20 intraperitoneally (i.p.). Micewere sacrificed if they developed either hind-limb paralysis orlost >20% body weight. The results are plotted as % survival vs. time(days). Median survival and long term survivors are shown.

FIG. 4. Relative dose-response curves generated using an MTSproliferation assay for Daudi cells, Raji cells and Ramos cells treatedwith a bispecific HID (DNL2—four hLL2 Fab fragments tethered to an hA20IgG) and a monospecific HID (Hex-hA20), compared with an hA20 IgGcontrol. In Daudi cells (top panel), DNL2 showed >100-fold and Hex-hA20showed >10,000 fold more potent antiproliferative activity than hA20IgG. In Raji cells (middle panel), Hex-hA20 displayed potentanti-proliferative activity, while DNL2 showed only minimal activity,compared to hA20 IgG. In Ramos cells (bottom panel), both DNLs andHex-hA20 displayed potent anti-proliferative activity compared to hA20IgG.

FIG. 5. In vitro cytotoxicity as determined by the MTS assay andapoptosis by the annexin V staining. Results shown are for Daudi (A),Raji (B), RL and DoHH2 (C), and the annexin V binding assay for Daudi(D). The concentrations of each primary antibody and GAH were 10 nM and10 μg/mL, respectively.

FIG. 6. Western blot analysis of proteins induced in Daudi by theparental antibodies and HexAbs. (A) Effect on phospho-Lyn, phospho-Syk,phosph-PLCγ2 and β-actin control of epratuzumab, rituximab, veltuzumab,20-20, 22-20, 20-22 and control anti-IgM. (B) Time course of effects ofphospho-Lyn and Lyn of 20-20, 22-20 and 20-22. (C) Effect onphospho-AKT, AKT and β-actin control of epratuzumab, rituximab,veltuzumab, 20-20, 22-20 and 20-22. (D) Effect on RKIP vs. β-actincontrol of epratuzumab, rituximab, veltuzumab, 20-20, 22-20 and 20-22.(E) Effect on phospho-Lyn and phospho-AKT vs. β-actin control ofepratuzumab, rituximab, veltuzumab, 20-20, 22-20 and 20-22. Parentantibodies used were each at 133 nM (A, C, D) or 10 nM (E). The HexAbsused were each at 10 nM (A-E); or anti-IgM, at 10 μg/mL (A, C). Thechanges in phospho-Lyn observed with 20-20, 22-20, and 20-22 within thefirst 24 hours are shown in (B).

FIG. 7. Modulation of ERK1/2 and p38 MAPK pathways. (A) Daudi cells weretreated with 133 nM each of rituximab, epratuzumab, and veltuzumab and10 nM each of 20-20, 22-20, and 20-22 separately for 24 hours. Cellswere immunoblotted and probed with phospho-specific antibodies as wellas with antibodies to ERK1/2, p38, and β-actin. Bar diagrams show therelative intensity of phospho-ERK or phospho-p38 induced by each agent,as determined by densitometry analysis of the results from 2 or moreindependent experiments. (B) Phospho-ERK1/2 induced by 20-20, 22-20, and20-22 or rituximab at 10 nM measured at various time points within thefirst 24 hours. (C) Up-regulation of phospho-ERK1/2 by crosslinkingrituximab and veltuzumab with GAH (10 μg/mL).

FIG. 8. Effects observed in Daudi cells. (A) Selective proteinspertaining to the NF-κB pathway, (B) selective proteins pertaining tothe Bcl-2 family, and (C) mitochondrial membrane depolarization.

FIG. 9. PTEN-PI3K pathway. (A) Time course of PTEN after eating Daudicells with 20-20, 22-20, and 20-22 at 10 nM and rituximab at 133 nM. Thesample corresponding to 20-22 at 24 hours was lost during loading andthus not analyzed. (B) Daudi cells transfected with PTEN siRNA showedreduced expression of PTEN, compared with the untreated cells or cellstransfected with control siRNA. (C) PTEN siRNA reduced the apoptosis ofDaudi by 20-20, 22-20, and 20-22 at 10 nM (P values with respect to*20-20, #22-20, and **20-22; P<0.05). (D) The PI3K inhibitor, LY294002(5 μM), enhanced the apoptosis induced by 20-20, 22-20, and 20-22 at 10nM, but not rituximab at 133 nM.

FIG. 10. Deregulation of cell cycle. (A) Histograms obtained from Daudicells treated with 100 nM of each agent, showing G₁ arrest induced by20-20, 22-20, and 20-22. (B) Comparison of cells in the G₁ phase aftertreatment with each agent at 10 or 100 nM. (C) Up-regulation of theCip/Kip family of proteins on treatment with 20-20, 20-22, and 22-20.(D) Down-regulation of cyclin D1 and p-Rb.

DETAILED DESCRIPTION Definitions

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

An “antibody” refers to a full-length (i.e., naturally occurring orformed by normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., antigen-binding) portion of an immunoglobulin molecule,like an antibody fragment.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, single domain antibodies (DABs or VHHs) andthe like, including half-molecules of IgG4 (van der Neut Kolfschoten etal. (Science 2007; 317(14 September):1554-1557). Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody. For example, an anti-CD20 antibodyfragment binds with an epitope of CD20. The term “antibody fragment”also includes isolated fragments consisting of the variable regions,such as the “Fv” fragments consisting of the variable regions of theheavy and light chains and recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“scFv proteins”).

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains. Additional FR amino acidsubstitutions from the parent, e.g. murine, antibody may be made. Theconstant domains of the antibody molecule are derived from those of ahuman antibody.

A “human antibody” is, for example, an antibody obtained from transgenicmice that have been genetically engineered to produce human antibodiesin response to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) forthe production of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includebut are not limited to antibodies, antibody fragments, drugs, toxins,enzymes, nucleases, hormones, immunomodulators, antisenseoligonucleotides, chelators, boron compounds, photoactive agents, dyesand radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes, contrast agents, fluorescent compoundsor molecules and enhancing agents (e.g., paramagnetic ions). Preferably,the diagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents, and fluorescent compounds.

An “immunoconjugate” is a conjugate of an antibody, antibody fragment,antibody fusion protein, bispecific antibody or multispecific antibodywith an atom, molecule, or a higher-ordered structure (e.g., with acarrier, a therapeutic agent, or a diagnostic agent). A “naked antibody”is an antibody that is not conjugated to any other agent.

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is covalently linked to another protein or peptide, such as thesame or different antibody or antibody fragment or a DDD or AD peptide.The fusion protein may comprise a single antibody component, amultivalent or multispecific combination of different antibodycomponents or multiple copies of the same antibody component. The fusionprotein may additionally comprise an antibody or an antibody fragmentand a therapeutic agent. Examples of therapeutic agents suitable forsuch fusion proteins include immunomodulators and toxins. One preferredtoxin comprises a ribonuclease (RNase), preferably a recombinant RNase.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity. For example, a diabody, where one binding site reacts withone antigen and the other with another antigen.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure.

Dock-and-Lock (DNL)

In preferred embodiments, multivalent monospecific or bispecificantibodies may be produced using the dock-and-lock (DNL) technology(see, e.g., U.S. Pat. Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787and 7,666,400; the Examples section of each of which is incorporatedherein by reference). The DNL method exploits specific protein/proteininteractions that occur between the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and the anchoring domain (AD) ofA-kinase anchoring proteins (AKAPs) (Baillie et al., FEBS Letters. 2005;579: 3264. Wong and Scott, Nat. Rev. Mol. Cell. Biol. 2004; 5: 959).PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,there are four types of PKA regulatory subunits—RIα, RIβ, RIIα and RIIβ.The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561).

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunit and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically. Using variouscombinations of linkers, adaptor modules and precursors, a wide varietyof DNL constructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

The skilled artisan will realize that the DNL technique may be utilizedto produce complexes comprising multiple copies of the same anti-CD20 oranti-CD22 antibody, or to attach one or more anti-CD20 antibodies to oneor more anti-CD22 antibodies, or to attach an anti-CD20 or anti-CD22antibody to an antibody that binds to a different antigen expressed byB-cells. Alternatively, the DNL technique may be used to attachantibodies to different effector moieties, such as toxins, cytokines,carrier proteins for siRNA and other known effectors.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL constructs, different AD or DDD sequences maybe utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 13) SHIQ1PPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 14) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 15) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 16)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 17) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 18) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 19) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 20)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 21) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 22) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 23) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Can et al., 2001, J Biol Chem 276:17332-38; Altoet al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al.,2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99;Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408, the entire text of each of which is incorporated herein byreference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:13 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 13) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:13are shown in Table 1. In devising Table 1, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. Even withsuch conservative substitutions, there are over twenty million possiblealternative sequences for the 44 residue peptide(2×3×2×2×2×2×2×2×2×2×2×2×2×2×2×4×2×2×2×2×2×4×2×4). A limited number ofsuch potential alternative DDD moiety sequences are shown in SEQ IDNO:24 to SEQ ID NO:43 below. The skilled artisan will realize that analmost unlimited number of alternative species within the genus of DDDmoieties can be constructed by standard techniques, for example using acommercial peptide synthesizer or well known site-directed mutagenesistechniques. The effect of the amino acid substitutions on AD moietybinding may also be readily determined by standard binding assays, forexample as disclosed in Alto et al. (2003, Proc Natl Acad Sci USA100:4445-50).

TABLE 1 Conservative Amino Acid Substitutions in DDD1(SEQ ID NO: 13). Consensus sequence disclosed as SEQ ID NO: 135. S  H  I Q   I  P P G  L  T E  L    L  Q G  Y  T  V   E  V   L   RT  K   N        A   S D      N A   S    D      K    R Q  Q P P  D  L   V E  F  A  V  E   Y   F  T R  L  R  E A R  AN  N      E     D   L   D      S K   K  D L K  L                    I                     I    I                    V                     V    V (SEQ ID NO: 24)THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 26)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 27)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 31)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 32)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 33)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 34)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 35)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 36)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 37)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 38)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 39)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 40)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 41)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 42)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 43)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:15), witha binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designedas a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:15 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 2 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:15), similar tothat shown for DDD1 (SEQ ID NO:13) in Table 1 above.

Even with such conservative substitutions, there are over thirty-fivethousand possible alternative sequences for the 17 residue AD1 (SEQ IDNO:15) peptide sequence (2×3×2×4×3×2×2×2×2×2×2×4). A limited number ofsuch potential alternative AD moiety sequences are shown in SEQ ID NO:44to SEQ ID NO:61 below. Again, a very large number of species within thegenus of possible AD moiety sequences could be made, tested and used bythe skilled artisan, based on the data of Alto et al. (2003). It isnoted that FIG. 2 of Alto (2003) shows an even large number of potentialamino acid substitutions that may be made, while retaining bindingactivity to DDD moieties, based on actual binding experiments.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 15)

TABLE 2 Conservative Amino Acid Substitutions in AD1(SEQ ID NO: 15). Consensus sequence disclosed as SEQ ID NO: 136.Q  I  E   Y  L   A   K  Q  I    V  D  N  A    I   Q  Q  AN  L  D   F  I     R  N      E  Q       N  N  L   V      T  V                                I          S                                   V NIEYLAKQIVDNAIQQA(SEQ ID NO: 44) QLEYLAKQIVDNAIQQA (SEQ ID NO: 45) QVEYLAKQIVDNAIQQA(SEQ ID NO: 46) QIDYLAKQIVDNAIQQA (SEQ ID NO: 47) QIEFLAKQIVDNAIQQA(SEQ ID NO: 48) QIETLAKQIVDNAIQQA (SEQ ID NO: 49) QIESLAKQIVDNAIQQA(SEQ ID NO: 50) QIEYIAKQIVDNAIQQA (SEQ ID NO: 51) QIEYVAKQIVDNAIQQA(SEQ ID NO: 52) QIEYLARQIVDNAIQQA (SEQ ID NO: 53) QIEYLAKNIVDNAIQQA(SEQ ID NO: 54) QIEYLAKQIVENAIQQA (SEQ ID NO: 55) QIEYLAKQIVDQAIQQA(SEQ ID NO: 56) QIEYLAKQIVDNAINQA (SEQ ID NO: 57) QIEYLAKQIVDNAIQNA(SEQ ID NO: 58) QIEYLAKQIVDNAIQQL (SEQ ID NO: 59) QIEYLAKQIVDNAIQQI(SEQ ID NO: 60) QIEYLAKQIVDNAIQQV (SEQ ID NO: 61)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:62),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:63-65.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 62)Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 63)QIEYHAKQIVDHAIHQA (SEQ ID NO: 64) QIEYVAKQIVDHAIHQA (SEQ ID NO: 65)

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL PLEYQAGLLVQNAIQQAI (SEQ ID NO: 66) AKAP79LLIETASSLVKNAIQLSI (SEQ ID NO: 67) AKAP-Lbc LIEEAASRIVDAVIEQVK(SEQ ID NO: 68) RI-Specific AKAPs AKAPce ALYQFADRFSELVISEAL(SEQ ID NO: 69) RIAD LEQVANQLADQIIKEAT (SEQ ID NO: 70) PV38FEELAWKIAKMIWSDVF (SEQ ID NO: 71) Dual-Specificity AKAPs AKAP7ELVRLSKRLVENAVLKAV (SEQ ID NO: 72) MAP2D TAEEVSARIVQVVTAEAV(SEQ ID NO: 73) DAKAP1 QIKQAAFQLISQVILEAT (SEQ ID NO: 74) DAKAP2LAWKIAKMIVSDVMQQ (SEQ ID NO: 75)

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:76-78. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:76), RIAD (SEQ IDNO:77) and PV-38 (SEQ ID NO:78). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 76) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 77) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 78)

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 3 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 3 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 15) AKAPIS-P QIEYLAKQIVDNAIQQA (SEQ ID NO: 79) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 80) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 81) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 82) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 83) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 84) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 85) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 86) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 87) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 88) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 89) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 90) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 91) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 92) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 93) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 94) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 95) Rab32-pepETSAKDNINTEEAARFLVEKILVNH (SEQ ID NO: 96)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:15). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 15)

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:13. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 13) SHIQ IP P GL TELL QGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:13) sequence, based on the data of Can et al. (2001) is shownin Table 4. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 1 andTable 2.

TABLE 4 Conservative Amino Acid Substitutions in DDD1(SEQ ID NO: 13). Consensus sequence disclosed as SEQ ID NO: 137. S H I Q

P

T E

Q

V

T N S I L A Q

P

V E

V E

T R

R E A

A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In various embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, Ile, val; Arg (R)gin, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (O) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, vat, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Preparation of Antibodies

The complexes described herein may comprise one or more monoclonalantibodies or fragments thereof. Rodent monoclonal antibodies tospecific antigens may be obtained by methods known to those skilled inthe art. (See, e.g., Kohler and Milstein, Nature 256: 495 (1975), andColigan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages2.5.1-2.6.7 (John Wiley & Sons 1991)).

General techniques for cloning murine immunoglobulin variable domainshave been disclosed, for example, by the publication of Orlandi et al.,Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for constructingchimeric antibodies are well known to those of skill in the art. As anexample, Leung et al., Hybridoma 13:469 (1994), disclose how theyproduced an LL2 chimera by combining DNA sequences encoding the V_(k)and V_(H) domains of LL2 monoclonal antibody, an anti-CD22 antibody,with respective human and IgG₁ constant region domains. This publicationalso provides the nucleotide sequences of the LL2 light and heavy chainvariable regions, V_(k) and V_(H), respectively. Techniques forproducing humanized antibodies are disclosed, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993).

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimericantibody with one or more different human FR. Specifically, mouse CDRsare transferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. (See, e.g., Tempest et al., Biotechnology 9:266 (1991)and Verhoeyen et al., Science 239: 1534 (1988)).

A fully human antibody can be obtained from a transgenic non-humananimal. (See, e.g., Mendez et al., Nature Genetics, 15: 146-156, 1997;U.S. Pat. No. 5,633,425.) Methods for producing fully human antibodiesusing either combinatorial approaches or transgenic animals transformedwith human immunoglobulin loci are known in the art (e.g., Mancini etal., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb.Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr.Opin. Pharmacol. 3:544-50; each incorporated herein by reference). Suchfully human antibodies are expected to exhibit even fewer side effectsthan chimeric or humanized antibodies and to function in vivo asessentially endogenous human antibodies. In certain embodiments, theclaimed methods and procedures may utilize human antibodies produced bysuch techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as a hematopoietic cancer (Dantas-Barbosa et al.,2005). The advantage to constructing human antibodies from a diseasedindividual is that the circulating antibody repertoire may be biasedtowards antibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22, incorporated herein by reference). The final Fabfragments were digested with restriction endonucleases and inserted intothe bacteriophage genome to make the phage display library. Suchlibraries may be screened by standard phage display methods. The skilledartisan will realize that this technique is exemplary only and any knownmethod for making and screening human antibodies or antibody fragmentsby phage display may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XENOMOUSE® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets have been deposited at theATCC and/or have published variable region sequences and are availablefor use in the claimed methods and compositions. See, e.g., U.S. Pat.Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,15; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Exemplary known antibodies that may be of use for therapy of cancer orautoimmune disease within the scope of the claimed methods andcompositions include, but are not limited to, LL1 (anti-CD74), LL2 andRFB4 (anti-CD22), RS7 (anti-epithelial glycoprotein-1 (EGP-1)), PAM4 andKC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA orCEACAM5, also known as CD66e)), Mu-9 (anti-colon-specific antigen-p),Immu-31 (an anti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 orHuJ591 (anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), hL243 (anti-HLA-DR), R1 (anti-IGF-1R), A20 (anti-CD20), A19(anti-CD19), MN-3 or MN-15 (anti-CEACAM6). Such antibodies are known inthe art (e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090;6,183,744; 6,306,393; 6,653,104; 6,730,300; 6,899,864; 6,926,893;6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004;7,282,567; 7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; andU.S. Patent Application Publ. No. 20040202666 (now abandoned);20050271671; and 20060193865; the Examples section of each incorporatedherein by reference.) Specific known antibodies of use include, but arenot limited to, hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No.7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No.7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.7,541,440), hR1 (U.S. patent application Ser. No. 12/689,336), hRS7(U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440),AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372, depositedas ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575). Other knownantibodies are disclosed, for example, in U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20040202666(now abandoned); 20050271671; and 20060193865. The text of each recitedpatent or application is incorporated herein by reference with respectto the Figures and Examples sections.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, scFv and the like. Otherantibody fragments include, but are not limited to, F(ab′)₂ fragmentswhich can be produced by pepsin digestion of the antibody molecule andFab′ fragments which can be generated by reducing disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to Ruin a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredisclosed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

An antibody fragment can be prepared by known methods, for example, asdisclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 andreferences contained therein. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967),and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

A single complementarity-determining region (CDR) is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. (See, e.g., Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Single domain antibodies may be obtained,for example, from camels, alpacas or llamas by standard immunizationtechniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yauet al., J Immunol Methods 281:161-75, 2003; Maass et al., J ImmunolMethods 324:13-25, 2007). They can have potent antigen-binding capacityand can interact with novel epitopes that are inaccessible toconventional V_(H)-V_(L) pairs. (Muyldermans et al., 2001). Alpaca serumIgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs)(Maass et al., 2007). Alpacas may be immunized with known antigens, suchas TNF-α, and single domain antibodies can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca antibody coding sequences have beenidentified and may be used to construct single domain phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

In certain embodiments, the sequences of antibodies or antibodyfragments, such as the Fc portions of antibodies, may be varied tooptimize their physiological characteristics, such as the half-life inserum. Methods of substituting amino acid sequences in proteins arewidely known in the art, such as by site-directed mutagenesis (e.g.Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed,1989). In preferred embodiments, the variation may involve the additionor removal of one or more glycosylation sites in the Fc sequence (e.g.,U.S. Pat. No. 6,254,868, the Examples section of which is incorporatedherein by reference). In other preferred embodiments, specific aminoacid substitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797).

Multispecific and Multivalent Antibodies

Various embodiments may concern use of multispecific and/or multivalentantibodies. For example, an anti-CD20 antibody or fragment thereof andan anti-CD22 antibody or fragment thereof may be joined together bymeans such as the dock-and-lock technique described above. Othercombinations of antibodies or fragments thereof may be utilized. Forexample, the anti-CD20 or anti-CD22 antibody could be combined withanother antibody against a different epitope of the same antigen, oralternatively with an antibody against another antigen, such as CD4,CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37,CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7,HM1.24, HLA-DR, an angiogenesis factor, tenascin, VEGF, P1GF, ED-Bfibronectin, an oncogene, an oncogene product, NCA 66a-d, necrosisantigens, Ii, IL-2, T101, TAC, IL-6, MUC-1, TRAIL-R1 (DR4) or TRAIL-R2(DR5).

Methods for producing bispecific antibodies include engineeredrecombinant antibodies which have additional cysteine residues so thatthey crosslink more strongly than the more common immunoglobulinisotypes. (See, e.g., FitzGerald et al, Protein Eng 10:1221-1225, 1997).Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. (See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997). A variety of bispecific antibodies can beproduced using molecular engineering. In one form, the bispecificantibody may consist of, for example, a scFv with a single binding sitefor one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bispecific antibody may consist of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen.

Immunoconjugates

In preferred embodiments, an antibody or antibody fragment in a DNLcomplex may be directly attached to one or more therapeutic agents toform an immunoconjugate. Therapeutic agents may be attached, for exampleto reduced SH groups and/or to carbohydrate side chains. A therapeuticagent can be attached at the hinge region of a reduced antibodycomponent via disulfide bond formation. Alternatively, such agents canbe attached using a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic agentcan be conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of theimmunoconjugate is an antibody fragment. However, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofa full length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and6,254,868, the Examples section of which is incorporated herein byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

An alternative method for attaching therapeutic agents to an antibody orfragment involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted akyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach therapeutic agents toantibodies in vitro.

The specificity of the click chemistry reaction may be used as asubstitute for the antibody-hapten binding interaction used inpretargeting with bispecific antibodies. In this alternative embodiment,the specific reactivity of e.g., cyclooctyne moieties for azide moietiesor alkyne moieties for nitrone moieties may be used in an in vivocycloaddition reaction. An antibody-based DNL complex is activated byincorporation of a substituted cyclooctyne, an azide or a nitronemoiety. A targetable construct is labeled with one or more diagnostic ortherapeutic agents and a complementary reactive moiety. I.e., where theantibody comprises a cyclooctyne, the targetable construct will comprisean azide; where the antibody comprises a nitrone, the targetableconstruct will comprise an alkyne, etc. The DNL complex comprising anactivated antibody or fragment is administered to a subject and allowedto localize to a targeted cell, tissue or pathogen, as disclosed forpretargeting protocols. The reactive labeled targetable construct isthen administered. Because the cyclooctyne, nitrone or azide on thetargetable construct is unreactive with endogenous biomolecules andhighly reactive with the complementary moiety on the antibody, thespecificity of the binding interaction results in the highly specificbinding of the targetable construct to the tissue-localized antibody.

Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially with the subject DNL complexes. For example, drugs,toxins, oligonucleotides, immunomodulators, hormones, hormoneantagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesisinhibitors, other antibodies or fragments thereof, etc. The therapeuticagents recited here are those agents that also are useful foradministration separately with an antibody or fragment thereof asdescribed above. Therapeutic agents include, for example, cytotoxicagents such as vinca alkaloids, anthracyclines, gemcitabine,epipodophyllotoxins, taxanes, antimetabolites, alkylating agents,antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti-angiogenic andpro-apoptotic agents, particularly doxorubicin, methotrexate, taxol,CPT-11, camptothecans, proteosome inhibitors, mTOR inhibitors, HDACinhibitors, tyrosine kinase inhibitors, and others.

Other useful cytotoxic agents include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins,hormones, and the like. Suitable cytotoxic agents are described inREMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well asrevised editions of these publications.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to an anti-CD20 or anti-CD22antibody, for example as disclosed in U.S. Pat. No. 7,591,994, theExamples section of which is incorporated herein by reference.

The therapeutic agent may be selected from the group consisting ofaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,cisplatinum, irinotecan (CPT-11), SN-38, carboplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, egestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes,paclitaxel, testosterone propionate, thalidomide, thioguanine, thiotepa,teniposide, topotecan, uracil mustard, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, ranpirnase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate. Other toxins include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al.,Cell 47:641 (1986), Goldenberg, Calif. —A Cancer Journal for Clinicians44:43 (1994), Sharkey and Goldenberg, Calif. —A Cancer Journal forClinicians 56:226 (2006). Additional toxins suitable for use are knownto those of skill in the art and are disclosed in U.S. Pat. No.6,077,499, the Examples section of which is incorporated herein byreference.

The therapeutic agent may be an enzyme selected from the groupconsisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

As used herein, the term “immunomodulator” includes cytokines,lymphokines, monokines, stem cell growth factors, lymphotoxins,hematopoietic factors, colony stimulating factors (CSF), interferons(IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, transforming growth factor (TGF), TGF-α, TGF-β, insulin-likegrowth factor (IGF), erythropoietin, thrombopoietin, tumor necrosisfactor (TNF), TNF-α, TNF-β, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, interleukin (IL), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, S1 factor,IL-1, IL-1cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21, IL-25,L1F, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, LT, andthe like.

Exemplary anti-angiogenic agents may include angiostatin, endostatin,vasculostatin, canstatin, maspin, anti-VEGF binding molecules,anti-placental growth factor binding molecules, or anti-vascular growthfactor binding molecules.

In certain embodiments, the DNL complex may comprise one or morechelating moieties, such as NOTA, DOTA, DTPA, TETA, Tscg-Cys, orTsca-Cys. In certain embodiments, the chelating moiety may form acomplex with a therapeutic or diagnostic cation, such as Group II, GroupIII, Group IV, Group V, transition, lanthanide or actinide metalcations, Tc, Re, Bi, Cu, As, Ag, Au, At, or Pb.

The antibody or fragment thereof may be administered as animmunoconjugate comprising one or more radioactive isotopes useful fortreating diseased tissue. Particularly useful therapeutic radionuclidesinclude, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe,⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclide preferably has a decayenergy in the range of 20 to 6,000 keV, preferably in the ranges 60 to200 keV for an Auger emitter, 100-2,500 keV for a beta emitter and4,000-6,000 keV for an alpha emitter. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Interference RNA

In certain preferred embodiments the therapeutic agent may be a siRNA orinterference RNA species. The siRNA, interference RNA or therapeuticgene may be attached to a carrier moiety that is conjugated to anantibody or fragment thereof. A variety of carrier moieties for siRNAhave been reported and any such known carrier may be incorporated into atherapeutic antibody for use. Non-limiting examples of carriers includeprotamine (Rossi, 2005, Nat Biotech 23:682-84; Song et al., 2005, NatBiotech 23:709-17); dendrimers such as PAMAM dendrimers (Pan et al.,2007, Cancer Res. 67:8156-8163); polyethylenimine (Schiffelers et al.,2004, Nucl Acids Res 32:e149); polypropyleneimine (Taratula et al.,2009, J Control Release 140:284-93); polylysine (Inoue et al., 2008, JControl Release 126:59-66); histidine-containing reducible polycations(Stevenson et al., 2008, J Control Release 130:46-56); histone H1protein (Haberland et al., 2009, Mol Biol Rep 26:1083-93); cationiccomb-type copolymers (Sato et al., 2007, J Control Release 122:209-16);polymeric micelles (U.S. Patent Application Publ. No. 20100121043); andchitosan-thiamine pyrophosphate (Rojanarata et al., 2008, Pharm Res25:2807-14). The skilled artisan will realize that in general,polycationic proteins or polymers are of use as siRNA carriers. Theskilled artisan will further realize that siRNA carriers can also beused to carry other oligonucleotide or nucleic acid species, such asanti-sense oligonucleotides or short DNA genes.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); K-ras (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachreferenced patent incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Minis Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL complexes.

Exemplary siRNA species known in the art are listed in Table 5. AlthoughsiRNA is delivered as a double-stranded molecule, for simplicity onlythe sense strand sequences are shown in Table 5.

TABLE 5 Exemplary siRNA Sequences Target Sequence SEQ ID NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 97 VEGF R2 AAGCTCAGCACACAGAAAGACSEQ ID NO: 98 CXCR4 UAAAAUCUUCCUGCCCACCdT SEQ ID NO: 99 dT CXCR4GGAAGCUGUUGGCUGAAAAdT SEQ ID NO: 100 dT PPARC1 AAGACCAGCCUCUUUGCCCAGSEQ ID NO: 101 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 102 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 103 E1A binding UGACACAGGCAGGCUUGACUUSEQ ID NO: 104 protein Plasminogen GGTGAAGAAGGGCGTCCAA SEQ ID NO: 105activator K-ras GATCCGTTGGAGCTGTTGGCG SEQ ID NO: 106TAGTTCAAGAGACTCGCCAAC AGCTCCAACTTTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAGSEQ ID NO: 107 Apolipo- AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 108 protein EApolipo- AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 109 protein E Bcl-XUAUGGAGCUGCAGAGGAUGdT SEQ ID NO: 110 dT Raf-1 TTTGAATATCTGTGCTGAGAASEQ ID NO: 111 CACAGTTCTCAGCACAGATAT TCTTTTT Heat shockAATGAGAAAAGCAAAAGGTGC SEQ ID NO: 112 transcrip- CCTGTCTC tion factor 2IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 113 ThioredoxinAUGACUGUCAGGAUGUUGCdT SEQ ID NO: 114 dT CD44 GAACGAAUCCUGAAGACAUCUSEQ ID NO: 115 MMP14 AAGCCTGGCTACAGCAATATG SEQ ID NO: 116 CCTGTCTCMAPKAPK2 UGACCAUCACCGAGUUUAUdT SEQ ID NO: 117 dT FGFR1AAGTCGGACGCAACAGAGAAA SEQ ID NO: 118 ERBB2 CUACCUUUCUACGGACGUGdTSEQ ID NO: 119 dT BCL2L1 CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 120 ABL1TTAUUCCUUCUUCGGGAAGUC SEQ ID NO: 121 CEACAM1 AACCTTCTGGAACCCGCCCACSEQ ID NO: 122 CD9 GAGCATCTTCGAGCAAGAA SEQ ID NO: 123 CD151CATGTGGCACCGTTTGCCT SEQ ID NO: 124 Caspase 8 AACTACCAGAAAGGTATACCTSEQ ID NO: 125 BRCA1 UCACAGUGUCCUUUAUGUAdT SEQ ID NO: 126 dT p53GCAUGAACCGGAGGCCCAUTT SEQ ID NO: 127 CEACAM6 CCGGACAGTTCCATGTATASEQ ID NO: 128

The skilled artisan will realize that Table 5 represents a very smallsampling of the total number of siRNA species known in the art, and thatany such known siRNA may be utilized in the claimed methods andcompositions.

Immunotoxins Comprising Ranpirnase (Rap)

Ribonucleases, in particular, Rap (Lee, Exp Opin Biol Ther 2008;8:813-27) and its more basic variant, amphinase (Ardelt et al., CurrPharm Biotechnol 2008:9:215-25), are potential anti-tumor agents (Leeand Raines, Biodrugs 2008; 22:53-8). Rap is a single-chain ribonucleaseof 104 amino acids originally isolated from the oocytes of Rana pipiens.Rap exhibits cytostatic and cytotoxic effects on a variety of tumor celllines in vitro, as well as antitumor activity in vivo. The amphibianribonuclease enters cells via receptor-mediated endocytosis and onceinternalized into the cytosol, selectively degrades tRNA, resulting ininhibition of protein synthesis and induction of apoptosis.

Rap has completed a randomized Phase IIIb clinical trial, which comparedthe effectiveness of Rap plus doxorubicin with that of doxorubicin alonein patients with unresectable malignant mesothelioma, with the interimanalysis showing that the MST for the combination was 12 months, whilethat of the monotherapy was 10 months (Mutti and Gaudino, Oncol Rev2008; 2:61-5). Rap can be administered repeatedly to patients without anuntoward immune response, with reversible renal toxicity reported to bedose-limiting (Mikulski et al., J Clin Oncol 2002; 20:274-81; Int JOncol 1993; 3:57-64).

Conjugation or fusion of Rap to a tumor-targeting antibody or antibodyfragment is a promising approach to enhance its potency, as firstdemonstrated for LL2-onconase (Newton et al., Blood 2001; 97:528-35), achemical conjugate comprising Rap and a murine anti-CD22 monoclonalantibody (MAb), and subsequently for 2L-Rap-hLL1-γ4P, a fusion proteincomprising Rap and a humanized anti-CD74 MAb (Stein et al., Blood 2004;104:3705-11). The method used to generate 2L-Rap-hLL1-γ4P allowed us todevelop a series of structurally similar immunotoxins, referred to ingeneral as 2L-Rap-X, all of which consist of two Rap molecules, eachconnected via a flexible linker to the N-terminus of one L chain of anantibody of interest (X). We have also generated another series ofimmunotoxins of the same design, referred to as 2LRap(Q)-X, bysubstituting Rap with its non-glycosylation form of Rap, designated asRap(Q) to denote that the potential glycosylation site at Asn69 ischanged to Gln (or Q, single letter code). For both series, we made theIgG as either IgG1(γ1) or IgG4(γ4), and to prevent the formation of IgG4half molecules (Aalberse and Schuurman, Immunology 2002; 105:9-19), weconverted the serine residue in the hinge region (S228) of IgG4 toproline (γ4P). A pyroglutamate residue at the N-terminus of Rap isrequired for the RNase to be fully functional (Liao et al., NucleicAcids Res 2003; 31:5247-55).

The skilled artisan will recognize that the cytotoxic RNase moietiessuitable for use in the present invention include polypeptides having anative ranpirnase structure and all enzymatically active variantsthereof. These molecules advantageously have an N-terminal pyroglutamicacid resides that appears essential for RNase activity and are notsubstantially inhibited by mammalian RNase inhibitors. Nucleic acid thatencodes a native cytotoxic RNase may be prepared by cloning andrestriction of appropriate sequences, or using DNA amplification withpolymerase chain reaction (PCR). The amino acid sequence of Rana Pipiensranpirnase can be obtained from Ardelt et al., J. Biol. Chem., 256: 245(1991), and cDNA sequences encoding native ranpirnase, or aconservatively modified variation thereof, can be gene-synthesized bymethods similar to the en bloc V-gene assembly method used in hLL2humanization. (Leung et al., Mol. Immunol., 32: 1413, 1995). Methods ofmaking cytotoxic RNase variants are known in the art and are within theskill of the routineer.

As described in the Examples below, Rap conjugates of targetingantibodies may be made using the DNL technology. The DNL Rap-antibodyconstructs show potent cytotoxic activity that can be targeted todisease-associated cells.

Diagnostic Agents

In various embodiments, the DNL complexes may be conjugated to, or maybind a targetable construct comprising one or more diagnostic agents.Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹⁸F,⁵²Fe, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y,⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As,⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, orpositron-emitters.

Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III).

Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds. A widevariety of fluorescent labels are known in the art, including but notlimited to fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

Methods of Therapeutic Treatment

The claimed methods and compositions are of use for treating diseasestates, such as B-cell lymphomas or leukemias, autoimmune disease orimmune system dysfunction (e.g., graft-versus-host disease). The methodsmay comprise administering a therapeutically effective amount of atherapeutic antibody or fragment thereof or an immunoconjugate, eitheralone or in conjunction with one or more other therapeutic agents,administered either concurrently or sequentially.

Multimodal therapies may include therapy with other antibodies, such asantibodies against CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, HM1.24, HLA-DR, an angiogenesis factor, tenascin,VEGF, P1GF, ED-B fibronectin, an oncogene, an oncogene product, NCA66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, MUC-1, TRAIL-R1(DR4) or TRAIL-R2 (DR5) in the form of naked antibodies, fusionproteins, or as immunoconjugates. Various antibodies of use are known tothose of skill in the art. See, for example, Ghetie et al., Cancer Res.48:2610 (1988); Hekman et al., Cancer Immunol. Immunother. 32:364(1991); Longo, Curr. Opin. Oncol. 8:353 (1996), U.S. Pat. Nos.5,798,554; 6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164;7,230,084; 7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655;7,312,318; 7,612,180; 7,501,498; the Examples section of each of whichis incorporated herein by reference.

In another form of multimodal therapy, subjects may receive therapeuticDNL complexes in conjunction with standard chemotherapy. For example,“CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and 150-200mg/m² carmustine) is a regimen used to treat non-Hodgkin's lymphoma.Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitablecombination chemotherapeutic regimens are well-known to those of skillin the art. See, for example, Freedman et al., “Non-Hodgkin'sLymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.(eds.), pages 2028-2068 (Lea & Febiger 1993). As an illustration, firstgeneration chemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

Therapeutic antibody complexes, such as DNL complexes, can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic antibody complex is combined in amixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The therapeutic antibody complex can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Preferably, the therapeutic antibody complex is infused over a period ofless than about 4 hours, and more preferably, over a period of less thanabout 3 hours. For example, the first 25-50 mg could be infused within30 minutes, preferably even 15 min, and the remainder infused over thenext 2-3 hrs. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The therapeutic antibody complex may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. Preferably, the therapeutic antibody complex is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours.

More generally, the dosage of an administered therapeutic antibodycomplex for humans will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. It may be desirable to provide the recipientwith a dosage of therapeutic antibody complex that is in the range offrom about 1 mg/kg to 25 mg/kg as a single intravenous infusion,although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy.

Alternatively, a therapeutic antibody complex may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, the therapeutic antibody complex may be administered twice per weekfor 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m²(340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kgpatient), it may be administered once or even twice weekly for 4 to 10weeks. Alternatively, the dosage schedule may be decreased, namely every2 or 3 weeks for 2-3 months. It has been determined, however, that evenhigher doses, such as 20 mg/kg once weekly or once every 2-3 weeks canbe administered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic immunoconjugate or naked antibody.Control release preparations can be prepared through the use of polymersto complex or adsorb the DNL complex. For example, biocompatiblepolymers include matrices of poly(ethylene-co-vinyl acetate) andmatrices of a polyanhydride copolymer of a stearic acid dimer andsebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rateof release of a DNL complex from such a matrix depends upon themolecular weight of the DNL complex, the amount of DNL complex withinthe matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

Cancer Therapy

In preferred embodiments, the DNL complexes are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastomamultiforme, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors,medullary thyroid cancer, differentiated thyroid carcinoma, breastcancer, ovarian cancer, colon cancer, rectal cancer, endometrial canceror uterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, aswell as head-and-neck cancer. The term “cancer” includes primarymalignant cells or tumors (e.g., those whose cells have not migrated tosites in the subject's body other than the site of the originalmalignancy or tumor) and secondary malignant cells or tumors (e.g.,those arising from metastasis, the migration of malignant cells or tumorcells to secondary sites that are different from the site of theoriginal tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Therapy of Autoimmune Disease

Anti-CD20 and/or anti-CD22 DNL complexes can be used to treat immunedysregulation disease and related autoimmune diseases. Immune diseasesmay include acute idiopathic thrombocytopenic purpura, Addison'sdisease, adult respiratory distress syndrome (ARDS), agranulocytosis,allergic conditions, allergic encephalomyelitis, allergic neuritis,amyotrophic lateral sclerosis (ALS), ankylosing spondylitis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, aplastic anemia,arthritis, asthma, atherosclerosis, autoimmune disease of the testis andovary, autoimmune endocrine diseases, autoimmune myocarditis, autoimmuneneutropenia, autoimmune polyendocrinopathies, autoimmune polyglandularsyndromes (or polyglandular endocrinopathy syndromes), autoimmunethrombocytopenia, Bechet disease, Berger's disease (IgA nephropathy),bronchiolitis obliterans (non-transplant), bullous pemphigoid,Castleman's syndrome, Celiac sprue (gluten enteropathy), central nervoussystem (CNS) inflammatory disorders, chronic active hepatitis, chronicidiopathic thrombocytopenic purpura dermatomyositis, colitis, conditionsinvolving infiltration of T cells and chronic inflammatory responses,coronary artery disease, Crohn's disease, cryoglobulinemia, dermatitis,dermatomyositis, diabetes mellitus, diseases involving leukocytediapedesis, eczema, encephalitis, erythema multiforme, erythema nodosum,Factor VIII deficiency, fibrosing alveolitis, giant cell arteritis,glomerulonephritis, Goodpasture's syndrome, graft versus host disease(GVHD), granulomatosis, Grave's disease, Guillain-Barre Syndrome,Hashimoto's thyroiditis, hemophilia A, Henoch-Schonlein purpura,idiopathic hypothyroidism, idiopathic thrombocytopenic purpura (ITP),IgA nephropathy, IgA nephropathy, IgM mediated neuropathy, immunecomplex nephritis, immune hemolytic anemia including autoimmunehemolytic anemia (AIHA), immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytes,immune-mediated thrombocytopenias, juvenile onset diabetes, juvenilerheumatoid arthritis, Lambert-Eaton Myasthenic Syndrome, large vesselvasculitis, leukocyte adhesion deficiency, leukopenia, lupus nephritis,lymphoid interstitial pneumonitis (HIV), medium vessel vasculitis,membranous nephropathy, meningitis, multiple organ injury syndrome,multiple sclerosis, myasthenia gravis, osteoarthritis, pancytopenia,pemphigoid bullous, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychondritis, polyglandular syndromes, polymyalgia,polymyositis, post-streptococcal nephritis, primary biliary cirrhosis,primary hypothyroidism, psoriasis, psoriatic arthritis, pure red cellaplasia (PRCA), rapidly progressive glomerulonephritis, Reiter'sdisease, respiratory distress syndrome, responses associated withinflammatory bowel disease, Reynaud's syndrome, rheumatic fever,rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,solid organ transplant rejection, Stevens-Johnson syndrome, stiff-mansyndrome, subacute thyroiditis, Sydenham's chorea, systemic lupuserythematosus (SLE), systemic scleroderma and sclerosis, tabes dorsalis,Takayasu's arteritis, thromboangitis obliterans, thromboticthrombocytopenic purpura (TTP), thyrotoxicosis, toxic epidermalnecrolysis, tuberculosis, Type I diabetes, ulcerative colitis, uveitis,vasculitis (including ANCA) and Wegener's granulomatosis.

Kits

Various embodiments may concern kits containing DNL constructs and/orother components. Such components may include a targetable construct foruse with such DNL complexes. In alternative embodiments it iscontemplated that a targetable construct may be attached to one or moredifferent therapeutic and/or diagnostic agents.

If the composition containing components for administration is notformulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused for certain applications.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

Example 1 Preparation of Dock-and-Lock (DNL) Constructs

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibody, antibody fragment,immunomodulator, cytokine, PEG moiety, toxin, antigen or xenoantigen orother effector moiety. For certain preferred embodiments, antibodies andcytokines may be produced as fusion proteins comprising either adimerization and docking domain (DDD) or anchoring domain (AD) sequence.Although in preferred embodiments the DDD and AD moieties may be joinedto antibodies, antibody fragments, cytokines, toxins or other effectormoieties as fusion proteins, the skilled artisan will realize that othermethods of conjugation exist, such as chemical cross-linking, clickchemistry reaction, etc.

The technique is not limiting and any protein or peptide of use may beproduced as an AD or DDD fusion protein for incorporation into a DNLconstruct. Where chemical cross-linking is utilized, the AD and DDDconjugates may comprise any molecule that may be cross-linked to an ADor DDD sequence using any cross-linking technique known in the art. Incertain exemplary embodiments, a dendrimer or other polymeric moietysuch as polyethyleneimine or polyethylene glycol (PEG), may beincorporated into a DNL construct, as described in further detail below.

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors.

To generate Fab-DDD expression vectors, the coding sequences for thehinge, CH2 and CH3 domains of the heavy chain were replaced with asequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and a DDD moiety, such as the first 44 residues of humanRIIα (referred to as DDD1, SEQ ID NO:13). To generate Fab-AD expressionvectors, the sequences for the hinge, CH2 and CH3 domains of IgG werereplaced with a sequence encoding the first 4 residues of the hinge, a15 residue Gly-Ser linker and an AD moiety, such as a 17 residuesynthetic AD called AKAP-IS (referred to as AD1, SEQ ID NO:15), whichwas generated using bioinformatics and peptide array technology andshown to bind RIIα dimers with a very high affinity (0.4 nM). See Alto,et al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC, SEQ ID NO:129) followed byfour glycines and a serine, with the final two codons (GS) comprising aBam HI restriction site. The 410 bp PCR amplifier was cloned into thePGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD1 preceded by 11 residues of the linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 130) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplifier was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 131)

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplifierwas cloned into the PGEMT® vector and screened for inserts in the 17(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective PGEMT® shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Production and Purification of h679-Fab-AD1

The h679-Fd-AD1-pdHL2 vector was linearized by digestion with Sal Irestriction endonuclease and transfected into Sp/EEE myeloma cells byelectroporation. The di-cistronic expression vector directs thesynthesis and secretion of both h679 kappa light chain and h679 Fd-AD1,which combine to form h679 Fab-AD1. Following electroporation, the cellswere plated in 96-well tissue culture plates and transfectant cloneswere selected with 0.05 μM methotrexate (MTX). Clones were screened forprotein expression by ELISA using microtiter plates coated with aBSA-IMP260 (HSG) conjugate and detection with HRP-conjugated goatanti-human Fab. BIAcore analysis using an HSG (IMP239) sensorchip wasused to determine the productivity by measuring the initial slopeobtained from injection of diluted media samples. The highest producingclone had an initial productivity of approximately 30 mg/L. A total of230 mg of h679-Fab-AD1 was purified from 4.5 liters of roller bottleculture by single-step IMP291 affinity chromatography. Culture media wasconcentrated approximately 10-fold by ultrafiltration before loadingonto an IMP291-affigel column. The column was washed to baseline withPBS and h679-Fab-AD1 was eluted with 1 M imidazole, 1 mM EDTA, 0.1 MNaAc, pH 4.5. SE-HPLC analysis of the eluate shows a single sharp peakwith a retention time consistent with a 50 kDa protein (not shown). Onlytwo bands, which represent the polypeptide constituents of h679-AD1,were evident by reducing SDS-PAGE analysis (not shown).

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

Construction of N-DDD1-Fd-hMN-14-dHL2

N-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinN-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the aminoterminus of V_(H) via a flexible peptide spacer. The expression vectorwas engineered as follows. The DDD1 domain was amplified by PCR.

As a result of the PCR, an NcoI restriction site and the coding sequencefor part of the linker containing a BamHI restriction were appended tothe 5′ and 3′ ends, respectively. The 170 bp PCR amplifier was clonedinto the PGEMT® vector and clones were screened for inserts in the T7(5′) orientation. The 194 bp insert was excised from the PGEMT® vectorwith NcoI and SalI restriction enzymes and cloned into the SV3 shuttlevector, which was prepared by digestion with those same enzymes, togenerate the intermediate vector DDD1-SV3.

The hMN-14 Fd sequence was amplified by PCR. As a result of the PCR, aBamHI restriction site and the coding sequence for part of the linkerwere appended to the 5′ end of the amplifier. A stop codon and EagIrestriction site was appended to the 3′ end. The 1043 bp amplifier wascloned into pGemT. The hMN-14-Fd insert was excised from PGEMT® withBamHI and EagI restriction enzymes and then ligated with DDD1-SV3vector, which was prepared by digestion with those same enzymes, togenerate the construct N-DDD1-hMN-14Fd-SV3.

The N-DDD1-hMN-14 Fd sequence was excised with XhoI and EagI restrictionenzymes and the 1.28 kb insert fragment was ligated with a vectorfragment that was prepared by digestion of C-hMN-14-pdHL2 with thosesame enzymes. The final expression vector was N-DDD1-Fd-hMN-14-pDHL2.The N-linked Fab fragment exhibited similar DNL complex formation andantigen binding characteristics as the C-linked Fab fragment (notshown).

Production and Purification of N-DDD1-Fab-hMN-14 and C-DDD1-Fab-hMN-14

The C-DDD1-Fd-hMN-14-pdHL2 and N-DDD1-Fd-hMN-14-pdHL2 vectors weretransfected into Sp2/0-derived myeloma cells by electroporation.C-DDD1-Fd-hMN-14-pdHL2 is a di-cistronic expression vector, whichdirects the synthesis and secretion of both hMN-14 kappa light chain andhMN-14 Fd-DDD1, which combine to form C-DDD1-hMN-14 Fab.N-DDD1-hMN-14-pdHL2 is a di-cistronic expression vector, which directsthe synthesis and secretion of both hMN-14 kappa light chain andN-DDD1-Fd-hMN-14, which combine to form N-DDD1-Fab-hMN-14. Each fusionprotein forms a stable homodimer via the interaction of the DDD1 domain.

Following electroporation, the cells were plated in 96-well tissueculture plates and transfectant clones were selected with 0.05 μMmethotrexate (MTX). Clones were screened for protein expression by ELISAusing microtiter plates coated with WI2 (a rat anti-id monoclonalantibody to hMN-14) and detection with HRP-conjugated goat anti-humanFab. The initial productivity of the highest producing C-DDD1-Fab-hMN14Fab and N-DDD1-Fab-hMN14 Fab clones was 60 mg/L and 6 mg/L,respectively.

Affinity Purification of N-DDD1-hMN-14 and C-DDD1-hMN-14 withAD1-Affigel

The DDD/AD interaction was utilized to affinity purify DDD1-containingconstructs. AD1-C is a peptide that was made synthetically consisting ofthe AD1 sequence and a carboxyl terminal cysteine residue, which wasused to couple the peptide to Affigel following reaction of thesulfhydryl group with chloroacetic anhydride. DDD-containing dimerstructures specifically bind to the AD1-C-Affigel resin at neutral pHand can be eluted at low pH (e.g., pH 2.5).

A total of 81 mg of C-DDD1-Fab-hMN-14 was purified from 1.2 liters ofroller bottle culture by single-step AD1-C affinity chromatography.Culture media was concentrated approximately 10-fold by ultrafiltrationbefore loading onto an AD1-C-affigel column. The column was washed tobaseline with PBS and C-DDD1-Fab-hMN-14 was eluted with 0.1 M Glycine,pH 2.5. SE-HPLC analysis of the eluate showed a single protein peak witha retention time consistent with a 107 kDa protein (not shown). Thepurify was also confirmed by reducing SDS-PAGE, showing only two bandsof molecular size expected for the two polypeptide constituents ofC-DDD1-Fab-hMN-14 (not shown).

A total of 10 mg of N-DDD1-hMN-14 was purified from 1.2 liters of rollerbottle culture by single-step AD1-C affinity chromatography as describedabove. SE-HPLC analysis of the eluate showed a single protein peak witha retention time similar to C-DDD1-Fab-hMN-14 and consistent with a 107kDa protein (not shown). Reducing SDS-PAGE showed only two bandsattributed to the polypeptide constituents of N-DDD1-Fab-hMN-14 (notshown).

The binding activity of C-DDD1-Fab-hMN-14 was determined by SE-HPLCanalysis of samples in which the test article was mixed with variousamounts of WI2. A sample prepared by mixing WI2 Fab andC-DDD1-Fab-hMN-14 at a molar ratio of 0.75:1 showed three peaks, whichwere attributed to unbound C-DDD1-Fab-hMN14 (8.71 min),C-DDD1-Fab-hMN-14 bound to one WI2 Fab (7.95 min), and C-DDD1-Fab-hMN14bound to two WI2 Fabs (7.37 min) (not shown). When a sample containingWI2 Fab and C-DDD1-Fab-hMN-14 at a molar ratio of 4 was analyzed, only asingle peak at 7.36 minutes was observed (not shown). These resultsdemonstrated that hMN14-Fab-DDD1 is dimeric and has two active bindingsites. Very similar results were obtained when this experiment wasrepeated with N-DDD1-Fab-hMN-14.

A competitive ELISA demonstrated that both C-DDD1-Fab-hMN-14 andN-DDD1-Fab-hMN-14 bind to CEA with an avidity similar to hMN-14 IgG, andsignificantly stronger than monovalent hMN-14 Fab (not shown). ELISAplates were coated with a fusion protein containing the epitope (A3B3)of CEA for which hMN-14 is specific.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 (SEQ ID NO:14) appended to the carboxyl terminus of theFd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker. Thefusion protein secreted is composed of two identical copies of hMN-14Fab held together by non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

N-DDD2-Fd-hMN-14-pdHL2

N-DDD2-hMN-14-pdHL2 is an expression vector for production ofN-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 (SEQ ID NO:14) appended to the amino terminus of theFd. The DDD2 is coupled to the V_(H) domain via a 15 amino acid residueGly/Ser peptide linker. DDD2 has a cysteine residue preceding thedimerization and docking sequences, which are identical to those ofDDD1. The fusion protein secreted is composed of two identical copies ofhMN-14 Fab held together by non-covalent interaction of the DDD2domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (DDD2 Top and DDD2 Bottom), whichcomprise residues 1-13 of DDD2, were made synthetically. Theoligonucleotides were annealed and phosphorylated with T4 polynucleotidekinase (PNK), resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases NcoI and PstI, respectively.

The duplex DNA was ligated with a vector fragment, DDD1-hMN14 Fd-SV3that was prepared by digestion with NcoI and PstI, to generate theintermediate construct DDD2-hMN14 Fd-SV3. A 1.28 kb insert fragment,which contained the coding sequence for DDD2-hMN14 Fd, was excised fromthe intermediate construct with XhoI and EagI restriction endonucleasesand ligated with hMN14-pdHL2 vector DNA that was prepared by digestionwith those same enzymes. The final expression vector isN-DDD2-Fd-hMN-14-pdHL2.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2 (SEQID NO:16) appended to the carboxyl terminal end of the CH1 domain via a14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteineresidue preceding and another one following the anchor domain sequenceof AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 2 Generation of TF1 DNL Construct

A large scale preparation of a DNL construct, referred to as TF1, wascarried out as follows. N-DDD2-Fab-hMN-14 (Protein L-purified) andh679-Fab-AD2 (IMP-291-purified) were first mixed in roughlystoichiometric concentrations in 1 mM EDTA, PBS, pH 7.4. Before theaddition of TCEP, SE-HPLC did not show any evidence of a₂b formation(not shown). Instead there were peaks representing a₄ (7.97 min; 200kDa), a₂ (8.91 min; 100 kDa) and B (10.01 min; 50 kDa). Addition of 5 mMTCEP rapidly resulted in the formation of the a₂b complex asdemonstrated by a new peak at 8.43 min, consistent with a 150 kDaprotein (not shown). Apparently there was excess B in this experiment asa peak attributed to h679-Fab-AD2 (9.72 min) was still evident yet noapparent peak corresponding to either a₂ or a₄ was observed. Afterreduction for one hour, the TCEP was removed by overnight dialysisagainst several changes of PBS. The resulting solution was brought to10% DMSO and held overnight at room temperature.

When analyzed by SE-HPLC, the peak representing a₂b appeared to besharper with a slight reduction of the retention time by 0.1 min to 8.31min (not shown), which, based on our previous findings, indicates anincrease in binding affinity. The complex was further purified byIMP-291 affinity chromatography to remove the kappa chain contaminants.As expected, the excess h679-AD2 was co-purified and later removed bypreparative SE-HPLC (not shown).

TF1 is a highly stable complex. When IN was tested for binding to an HSG(IMP-239) sensorchip, there was no apparent decrease of the observedresponse at the end of sample injection. In contrast, when a solutioncontaining an equimolar mixture of both C-DDD1-Fab-hMN-14 andh679-Fab-AD1 was tested under similar conditions, the observed increasein response units was accompanied by a detectable drop during andimmediately after sample injection, indicating that the initially formeda₂b structure was unstable. Moreover, whereas subsequent injection ofWI2 gave a substantial increase in response units for TF1, no increasewas evident for the C-DDD1/AD1 mixture.

The additional increase of response units resulting from the binding ofWI2 to TF1 immobilized on the sensorchip corresponds to two fullyfunctional binding sites, each contributed by one subunit ofN-DDD2-Fab-hMN14. This was confirmed by the ability of TF1 to bind twoFab fragments of WI2 (not shown). When a mixture containing h679-AD2 andN-DDD1-hMN14, which had been reduced and oxidized exactly as TF1, wasanalyzed by BIAcore, there was little additional binding of WI2 (notshown), indicating that a disulfide-stabilized a₂b complex such as TF1could only form through the interaction of DDD2 and AD2.

Two improvements to the process were implemented to reduce the time andefficiency of the process. First, a slight molar excess ofN-DDD2-Fab-hMN-14 present as a mixture of a₄/a₂ structures was used toreact with h679-Fab-AD2 so that no free h679-Fab-AD2 remained and anya₄/a₂ structures not tethered to h679-Fab-AD2, as well as light chains,would be removed by IMP-291 affinity chromatography. Second, hydrophobicinteraction chromatography (HIC) has replaced dialysis or diafiltrationas a means to remove TCEP following reduction, which would not onlyshorten the process time but also add a potential viral removing step.N-DDD2-Fab-hMN-14 and 679-Fab-AD2 were mixed and reduced with 5 mM TCEPfor 1 hour at room temperature. The solution was brought to 0.75 Mammonium sulfate and then loaded onto a Butyl FF HIC column. The columnwas washed with 0.75 M ammonium sulfate, 5 mM EDTA, PBS to remove TCEP.The reduced proteins were eluted from the HIC column with PBS andbrought to 10% DMSO. Following incubation at room temperature overnight,highly purified TF1 was isolated by IMP-291 affinity chromatography (notshown). No additional purification steps, such as gel filtration, wererequired.

Example 3 Generation of TF2 DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 4 Production of TF10 DNL Construct

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The TF10 bispecific ([hPAM4]₂×h679) antibody was producedusing the method disclosed for production of the (anti CEA)₂× anti HSGbsAb TF2, as described above. The TF10 construct bears two humanizedPAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD2. The reaction mixture was incubated at room temperaturefor 24 hours under mild reducing conditions using 1 mM reducedglutathione. Following reduction, the DNL reaction was completed by mildoxidation using 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP291-affigel resin, which binds with highspecificity to the h679 Fab.

Example 5 Serum Stability of TF1 and TF2

TF1 and TF2 were designed to be DNL complexes that could be used in vivowhere extensive dilution in blood and tissues would occur. The stabilityof TF2 in human sera was assessed using BIACORE. TF2 was diluted to 0.1mg/ml in fresh human serum, which was pooled from four donors, andincubated at 37° C. under 5% CO₂ for seven days. Daily samples werediluted 1:25 and then analyzed by BIACORE using an IMP239 HSGsensorchip. An injection of WI2 IgG was used to quantify the amount ofintact and fully active TF2. Serum samples were compared to controlsamples that were diluted directly from the stock. TF2 was highly stablein serum, retaining 98% of its bispecific binding activity after 7 days(not shown). Similar results were obtained for TF1 in either human ormouse serum (not shown).

Example 6 Biodistribution of TF2 in Tumor-Bearing Mice

Biodistribution studies were performed for TF2 in female athymic nudemice bearing s.c. human colorectal adenocarcinoma xenografts (LS 174T).Cells were expanded in tissue culture until enough cells had been grownto inject 50 mice s.c. with 1×10⁷ cells per mouse. After one week,tumors were measured and mice assigned to groups of 5 mice pertime-point. The mean tumor size at the start of this study was0.141±0.044 cm³. All the mice were injected with 40 μg ¹²⁵I-TF2 (250pmoles, 2 μCi). They were then sacrificed and necropsied at 0.5, 2, 4,16, 24, 48, and 72 hrs post-injection. A total of 35 mice were used inthis study. Tumor as well as various tissues were removed and placed ina gamma-counter to determine percent-injected dose per gram (% ID/g) intissue at each time-point.

Radioiodination of ¹²⁵I-TF2 resulted in 2.7% unbound isotope with aspecific activity of 1.48 mCi/mg. The labeled sample was then subjectedto SE-HPLC alone and after mixing with a 20-fold molar excess of CEA.Approximately 83% of the TF2 eluted off with a retention time of 10.1minutes (not shown). There was 9% aggregated material (RT=9.03 min) and8% low molecular weight material (RT=14.37 min) in the labeled TF2 (notshown). When mixed with CEA, 95% of the labeled TF2 shifted to a highmolecular weigh species (RT=7.25 min) (not shown). These resultsindicated that the labeled preparation was acceptable for administrationto the tumor-bearing mice.

Peak tumor uptake occurred at 4 h post-injection (10.3.+−.2.1% ID/g).Between 16 and 24 h post-injection, the amount of TF2 in the tumor isnot significantly different (5.3±1.1% ID/g and 5.37±0.7% ID/g),indicating that peptide could be administered anytime between these twotime-points, depending on blood values, without impacting tumortargeting. Uptake and clearance of TF2 from normal tissues was verysimilar to what has been observed previously for TF1. Both TF1 and TF2appeared to favor clearance through the RES system (spleen and liver).

Example 7 Production of AD- and DDD-linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Examples, the IgG andFab fusion proteins shown in Table 6 were constructed and incorporatedinto DNL constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 6 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 8 Antibody-Dendrimer DNL Complex for siRNA

Cationic polymers, such as polylysine, polyethylenimine, orpolyamidoamine (PAMAM)-based dendrimers, form complexes with nucleicacids. However, their potential applications as non-viral vectors fordelivering therapeutic genes or siRNAs remain a challenge. One approachto improve selectivity and potency of a dendrimeric nanoparticle may beachieved by conjugation with an antibody that internalizes upon bindingto target cells.

We synthesized and characterized a novel immunoconjugate, designatedE1-G5/2, which was made by the DNL method to comprise half of ageneration 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to astabilized dimer of Fab derived from hRS7, a humanized antibody that israpidly internalized upon binding to the Trop-2 antigen expressed onvarious solid cancers.

Methods

E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2and hRS7-Fab-DDD2, under mild redox conditions, followed by purificationon a Protein L column. To make AD2-G5/2, we derivatized the AD2 peptidewith a maleimide group to react with the single thiol generated fromreducing a G5 PAMAM with a cystamine core and used reversed-phase HPLCto isolate AD2-G5/2. We produced hRS7-Fab-DDD2 as a fusion protein inmyeloma cells, as described in the Examples above.

The molecular size, purity and composition of E1-G5/2 were analyzed bysize-exclusion HPLC, SDS-PAGE, and Western blotting. The biologicalfunctions of E1-G5/2 were assessed by binding to an anti-idiotypeantibody against hRS7, a gel retardation assay, and a DNase protectionassay.

Results

E1-G5/2 was shown by size-exclusion HPLC to consist of a major peak(>90%) flanked by several minor peaks (not shown). The threeconstituents of E1-G5/2 (Fd-DDD2, the light chain, and AD2-G5/2) weredetected by reducing SDS-PAGE and confirmed by Western blotting (notshown). Anti-idiotype binding analysis revealed E1-G5/2 contained apopulation of antibody-dendrimer conjugates of different size, all ofwhich were capable of recognizing the anti-idiotype antibody, thussuggesting structural variability in the size of the purchased G5dendrimer (not shown). Gel retardation assays showed E1-G5/2 was able tomaximally condense plasmid DNA at a charge ratio of 6:1 (+/−), with theresulting dendriplexes completely protecting the complexed DNA fromdegradation by DNase I (not shown).

Conclusion

The DNL technique can be used to build dendrimer-based nanoparticlesthat are targetable with antibodies. Such agents have improvedproperties as carriers of drugs, plasmids or siRNAs for applications invitro and in vivo. In preferred embodiments, anti-B-cell antibodies,such as anti-CD20 and/or anti-CD22, may be utilized to deliver cytotoxicor cytostatic siRNA species to targeted B-cells for therapy of lymphoma,leukemia, autoimmune or other diseases and conditions.

Example 9 Maleimide AD2 Conjugate for DNL Dendrimers

The peptide IMP 498 up to and including the PEG moiety was synthesizedon a Protein Technologies PS3 peptide synthesizer by the Fmoc method onSieber Amide resin (0.1 mmol scale). The maleimide was added manually bymixing the β-maleimidopropionic acid NHS ester withdiisopropylethylamine and DMF with the resin for 4 hr. The peptide wascleaved from the resin with 15 mL TFA, 0.5 mL H₂O, 0.5 mLtriisopropylsilane, and 0.5 mL thioanisole for 3 hr at room temperature.The peptide was purified by reverse phase HPLC using H₂O/CH₃CN TFAbuffers to obtain about 90 mg of purified product after lyophilization.

Synthesis of Reduced G5 Dendrimer (G5/2)

The G-5 dendrimer (10% in MeOH, Dendritic Nanotechnologies), 2.03 g,7.03×10⁻⁶ mol was reduced with 0.1426 TCEP.HCl 1:1 MeOH/H₂O (˜4 mL) andstirred overnight at room temperature. The reaction mixture was purifiedby reverse phase HPLC on a C-18 column eluted with 0.1% TFA H₂O/CH₃CNbuffers to obtain 0.0633 g of the desired product after lyophilization.

Synthesis of G5/2 Dendrimer-AD2 Conjugate

The G5/2 Dendrimer, 0.0469 g (3.35×10⁻⁶ mol) was mixed with 0.0124 g ofIMP 498 (4.4×10⁻⁶ mol) and dissolved in 1:1 MeOH/1M NaHCO₃ and mixed for19 hr at room temperature followed by treatment with 0.0751 gdithiothreitol and 0.0441 g TCEP.HCl. The solution was mixed overnightat room temperature and purified on a C4 reverse phase HPLC column using0.1% TFA H₂O/CH₃CN buffers to obtain 0.0033 g of material containing theconjugated AD2 and dendrimer as judged by gel electrophoresis andWestern blot.

Example 10 Targeted Delivery of siRNA Using Protamine Linked Antibodies

Summary

RNA interference (RNAi) has been shown to down-regulate the expressionof various proteins such as HER2, VEGF, Raf-1, bcl-2, EGFR and numerousothers in preclinical studies. Despite the potential of RNAi to silencespecific genes, the full therapeutic potential of RNAi remains to berealized due to the lack of an effective delivery system to target cellsin vivo.

To address this critical need, we developed novel DNL constructs havingmultiple copies of human protamine tethered to a tumor-targeting,internalizing hRS7 (anti-Trop-2) antibody for targeted delivery ofsiRNAs in vivo. A DDD2-L-thP1 module comprising truncated humanprotamine (thP1, residues 8 to 29 of human protamine 1) was produced, inwhich the sequences of DDD2 and thP1 were fused respectively to the N-and C-terminal ends of a humanized antibody light chain (not shown). Thesequence of the truncated hP1 (thP1) is shown below. Reaction ofDDD2-L-thP1 with the antibody hRS7-IgG-AD2 under mild redox conditions,as described in the Examples above, resulted in the formation of anE1-L-thP1 complex (not shown), comprising four copies of thP1 attachedto the carboxyl termini of the hRS7 heavy chains.

tHP1 RSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO: 133)

The purity and molecular integrity of E1-L-thP1 following Protein Apurification were determined by size-exclusion HPLC and SDS-PAGE (notshown). In addition, the ability of E1-L-thP1 to bind plasmid DNA orsiRNA was demonstrated by the gel shift assay (not shown). E1-L-thP1 waseffective at binding short double-stranded oligonucleotides (not shown)and in protecting bound DNA from digestion by nucleases added to thesample or present in serum (not shown).

The ability of the E1-L-thP1 construct to internalize siRNAs intoTrop-2-expressing cancer cells was confirmed by fluorescence microscopyusing FITC-conjugated siRNA and the human Calu-3 lung cancer cell line(not shown).

Methods

The DNL technique was employed to generate E1-L-thP1. The hRS7 IgG-ADmodule, constructed as described in the Examples above, was expressed inmyeloma cells and purified from the culture supernatant using Protein Aaffinity chromatography. The DDD2-L-thP1 module was expressed as afusion protein in myeloma cells and was purified by Protein L affinitychromatography. Since the CH3-AD2-IgG module possesses two AD2 peptidesand each can bind to a DDD2 dimer, with each DDD2 monomer attached to aprotamine moiety, the resulting E1-L-thP1 conjugate comprises fourprotamine groups. E1-L-thp1 was formed in nearly quantitative yield fromthe constituent modules and was purified to near homogeneity (not shown)with Protein A.

DDD2-L-thP1 was purified using Protein L affinity chromatography andassessed by size exclusion HPLC analysis and SDS-PAGE under reducing andnonreducing conditions (data not shown). A major peak was observed at9.6 min (not shown). SDS-PAGE showed a major band between 30 and 40 kDain reducing gel and a major band about 60 kDa (indicating a dimeric formof DDD2-L-thP1) in nonreducing gel (not shown). The results of Westernblotting confirmed the presence of monomeric DDD2-L-tP1 and dimericDDD2-L-tP1 on probing with anti-DDD antibodies (not shown).

To prepare the E1-L-thP1, hRS7-IgG-AD2 and DDD2-L-thP1 were combined inapproximately equal amounts and reduced glutathione (final concentration1 mM) was added. Following an overnight incubation at room temperature,oxidized glutathione was added (final concentration 2 mM) and theincubation continued for another 24 h. E1-L-thP1 was purified from thereaction mixture by Protein A column chromatography and eluted with 0.1M sodium citrate buffer (pH 3.5). The product peak (not shown) wasneutralized, concentrated, dialyzed with PBS, filtered, and stored inPBS containing 5% glycerol at 2 to 8° C. The composition of E1-L-thP1was confirmed by reducing SDS-PAGE (not shown), which showed thepresence of all three constituents (AD2-appended heavy chain,DDD2-L-htP1, and light chain).

The ability of DDD2-L-thP1 and E1-L-thP1 to bind DNA was evaluated bygel shift assay. DDD2-L-thP1 retarded the mobility of 500 ng of a linearform of 3-kb DNA fragment in 1% agarose at a molar ratio of 6 or higher(not shown). E1-L-thP1 retarded the mobility of 250 ng of a linear200-bp DNA duplex in 2% agarose at a molar ratio of 4 or higher (notshown), whereas no such effect was observed for hRS7-IgG-AD2 alone (notshown). The ability of E1-L-thP1 to protect bound DNA from degradationby exogenous DNase and serum nucleases was also demonstrated (notshown).

The ability of E1-L-thP1 to promote internalization of bound siRNA wasexamined in the Trop-2 expressing ME-180 cervical cell line (not shown).Internalization of the E1-L-thP1 complex was monitored using FITCconjugated goat anti-human antibodies. The cells alone showed nofluorescence (not shown). Addition of FITC-labeled siRNA alone resultedin minimal internalization of the siRNA (not shown). Internalization ofE1-L-thP1 alone was observed in 60 minutes at 37° C. (not shown).E1-L-thP1 was able to effectively promote internalization of boundFITC-conjugated siRNA (not shown). E1-L-thP1 (10 μg) was mixed withFITC-siRNA (300 nM) and allowed to form E1-L-thP1-siRNA complexes whichwere then added to Trop-2-expressing Calu-3 cells. After incubation for4 h at 37° C. the cells were checked for internalization of siRNA byfluorescence microscopy (not shown).

The ability of E1-L-thP1 to induce apoptosis by internalization of siRNAwas examined. E1-L-thP1 (10 μg) was mixed with varying amounts of siRNA(AllStars Cell Death siRNA, Qiagen, Valencia, Calif.). TheE1-L-thP1-siRNA complex was added to ME-180 cells. After 72 h ofincubation, cells were trypsinized and annexin V staining was performedto evaluate apoptosis. The Cell Death siRNA alone or E1-L-thP1 alone hadno effect on apoptosis (not shown). Addition of increasing amounts ofE1-L-thP1-siRNA produced a dose-dependent increase in apoptosis (notshown). These results show that E1-L-thP1 could effectively deliversiRNA molecules into the cells and induce apoptosis of target cells.

Conclusions

The DNL technology provides a modular approach to efficiently tethermultiple protamine molecules to the anti-Trop-2 hRS7 antibody resultingin the novel molecule E1-L-thP1. SDS-PAGE demonstrated the homogeneityand purity of E1-L-thP1. DNase protection and gel shift assays showedthe DNA binding activity of E1-L-thP1. E1-L-thP1 internalized in thecells like the parental hRS7 antibody and was able to effectivelyinternalize siRNA molecules into Trop-2-expressing cells, such as ME-180and Calu-3.

The skilled artisan will realize that the DNL technique is not limitedto any specific antibody or siRNA species. Rather, the same methods andcompositions demonstrated herein can be used to make targeted deliverycomplexes comprising any antibody (e.g., anti-CD20 or anti-CD22), anysiRNA carrier and any siRNA species. The use of a bivalent IgG intargeted delivery complexes would result in prolonged circulatinghalf-life and higher binding avidity to target cells, resulting inincreased uptake and improved efficacy.

Example 11 Ribonuclease Based DNL Immunotoxins Comprising QuadrupleRanpirnase (Rap) Conjugated to B-Cell Targeting Antibodies

We applied the DNL method to generate a novel class of immunotoxins,each of which comprises four copies of Rap site-specifically linked to abivalent IgG. We combined a recombinant Rap-DDD module, produced in E.coli, with recombinant, humanized IgG-AD modules, which were produced inmyeloma cells and targeted B-cell lymphomas and leukemias via binding toCD20 (hA20, veltuzumab), CD22 (hLL2, epratuzumab) or HLA-DR (hL243,IMMU-114), to generate 20-Rap, 22-Rap and C2-Rap, respectively. For eachconstruct, a dimer of Rap was covalently tethered to the C-terminus ofeach heavy chain of the respective IgG. A control construct, 14-Rap, wasmade similarly, using labetuzumab (hMN-14), that binds to an antigen(CEACAM5) not expressed on B-cell lymphomas/leukemias.

Rap-DDD2 (SEQ ID NO: 134)pQDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAP VHFVGVGSC GGGGSLECGHIQIPPGLTELLQGYTVEVLRQQPPDLVEF AVEYFTRLREARA VEHHHHHH

The deduced amino acid sequence of secreted Rap-DDD2 is shown above (SEQID NO:134). Rap, underlined; linker, italics; DDD2, bold; pQ,amino-terminal glutamine converted to pyroglutamate. Rap-DDD2 wasproduced in E. coli as inclusion bodies, which were purified by IMACunder denaturing conditions, refolded and then dialyzed into PBS beforepurification by Q-SEPHAROSE® anion exchange chromatography. SDS-PAGEunder reducing conditions resolved a protein band with a Mr appropriatefor Rap-DDD2 (18.6 kDa) (not shown). The final yield of purifiedRap-DDD2 was 10 mg/L of culture.

The DNL method was employed to rapidly generate a panel of IgG-Rapconjugates. The IgG-AD modules were expressed in myeloma cells andpurified from the culture supernatant using Protein A affinitychromatography. The Rap-DDD2 module was produced and mixed with IgG-AD2to form a DNL complex. Since the CH3-AD2-IgG modules possess two AD2peptides and each can tether a Rap dimer, the resulting IgG-Rap DNLconstruct comprises four Rap groups and one IgG. IgG-Rap is formednearly quantitatively from the constituent modules and purified to nearhomogeneity with Protein A.

Prior to the DNL reaction, the CH3-AD2-IgG exists as both a monomer, anda disulfide-linked dimer (not shown). Under non-reducing conditions, theIgG-Rap resolves as a cluster of high molecular weight bands of theexpected size between those for monomeric and dimeric CH3-AD2-IgG (notshown). Reducing conditions, which reduces the conjugates to theirconstituent polypeptides, shows the purity of the IgG-Rap and theconsistency of the DNL method, as only bands representingheavy-chain-AD2 (HC-AD2), kappa light chain and Rap-DDD2 were visualized(not shown).

Reversed phase HPLC analysis of 22-Rap (not shown) resolved a singleprotein peak at 9.10 min eluting between the two peaks ofCH3-AD2-IgG-hLL2, representing the monomeric (7.55 min) and the dimeric(8.00 min) forms. The Rap-DDD2 module was isolated as a mixture of dimerand tetramer (reduced to dimer during DNL), which were eluted at 9.30and 9.55 min, respectively (not shown).

LC/MS analysis of 22-Rap was accomplished by coupling reversed phaseHPLC using a C8 column with ESI-TOF mass spectrometry (not shown). Thespectrum of unmodified 22-Rap identifies two major species, havingeither two G0F (G0F/G0F) or one G0F plus one G1F (G0F/G1F) N-linkedglycans, in addition to some minor glycoforms (not shown). Enzymaticdeglycosylation resulted in a single deconvoluted mass consistent withthe calculated mass of 22-Rap (not shown). The resulting spectrumfollowing reduction with TCEP identified the heavy chain-AD2 polypeptidemodified with an N-linked glycan of the G0F or G1F structure as well asadditional minor forms (not shown). Each of the three subunitpolypeptides comprising 22-Rap were identified in the deconvolutedspectrum of the reduced and deglycosylated sample (not shown). Theresults confirm that both the Rap-DDD2 and HC-AD2 polypeptides have anamino terminal glutamine that is converted to pyroglutamate (pQ);therefore, 22-Rap has 6 of its 8 constituent polypeptides modified bypQ.

In vitro cytotoxicity was evaluated in three NHL cell lines. Each cellline expresses CD20 at a considerably higher surface density compared toCD22; however, the internalization rate for hLL2 (anti-CD22) is muchfaster than hA20 (anti-CD20). 14-Rap shares the same structure as 22-Rapand 20-Rap, but its antigen (CEACAM5) is not expressed by the NHL cells.Cells were treated continuously with IgG-Rap as single agents or withcombinations of the parental MAbs plus rRap. Both 20-Rap and 22-Rapkilled each cell line at concentrations above 1 nM, indicating thattheir action is cytotoxic as opposed to merely cytostatic (not shown).20-Rap was the most potent IgG-Rap, suggesting that antigen density maybe more important than internalization rate. Similar results wereobtained for Daudi and Ramos, where 20-Rap (EC50˜0.1 nM) was 3-6-foldmore potent than 22-Rap (not shown). The rituximab-resistant mantle celllymphoma line, Jeko-1, exhibits increased CD20 but decreased CD22,compared to Daudi and Ramos. Importantly, 20-Rap exhibited very potentcytotoxicity (EC₅₀˜20 pM) in Jeko-1, which was 25-fold more potent than22-Rap (not shown).

The DNL method provides a modular approach to efficiently tethermultiple cytotoxins onto a targeting antibody, resulting in novelimmunotoxins that are expected to show higher in vivo potency due toimproved pharmacokinetics and targeting specificity. LC/MS, RP-HPLC andSDS-PAGE demonstrated the homogeneity and purity of IgG-Rap. TargetingRap with a MAb to a cell surface antigen enhanced its tumor-specificcytotoxicity. Antigen density and internalization rate are both criticalfactors for the observed in vitro potency of IgG-Rap. In vitro resultsshow that CD20-, CD22-, or HLA-DR-targeted IgG-Rap have potent biologicactivity for therapy of B-cell lymphomas and leukemias.

Example 12 Production and Use of a DNL Construct Comprising TwoDifferent Antibody Moieties and a Cytokine

In certain embodiments, trimeric DNL constructs may comprise threedifferent effector moieties, for example two different antibody moietiesand a cytokine moiety. We report here the generation andcharacterization of the first bispecific MAb-IFNα, designated 20-C2-2b,which comprises two copies of IFN-α2b and a stabilized F(ab)₂ of hL243(humanized anti-HLA-DR; IMMU-114) site-specifically linked to veltuzumab(humanized anti-CD20). In vitro, 20-C2-2b inhibited each of fourlymphoma and eight myeloma cell lines, and was more effective thanmonospecific CD20-targeted MAb-IFNα or a mixture comprising the parentalantibodies and IFNα in all but one (HLA-DR⁻/CD20⁻) myeloma line (notshown), suggesting that 20-C2-2b should be useful in the treatment ofvarious hematopoietic disorders. The 20-C2-2b displayed greatercytotoxicity against KMS12-BM (CD20⁺/HLA-DR⁺myeloma) than monospecificMAb-IFNα that targets only HLA-DR or CD20 (not shown), indicating thatall three components in 20-C2-2b can contribute to toxicity. Ourfindings indicate that a given cell's responsiveness to MAb-IFNα dependson its sensitivity to IFNα and the specific antibodies, as well as theexpression and density of the targeted antigens.

Because 20-C2-2b has antibody-dependent cellular cytotoxicity (ADCC),but not CDC, and can target both CD20 and HLA-DR, it is useful fortherapy of a broad range of hematopoietic disorders that express eitheror both antigens.

Antibodies

The abbreviations used in the following discussion are: 20(C_(H)3-AD2-IgG-v-mab, anti-CD20 IgG DNL module); C2(C_(H)1-DDD2-Fab-hL243, anti-HLA-DR Fab₂ DNL module); 2b (dimericIFNα2B-DDD2 DNL module); 734 (anti-in-DTPA IgG DNL module used asnon-targeting control). The following MAbs were provided byImmunomedics, Inc.: veltuzumab or v-mab (anti-CD20 IgG₁), hL243γ4p(Immu-114, anti-HLA-DR IgG₄), a murine anti-IFNα MAb, and ratanti-idiotype MAbs to v-mab (WR2) and hL243 (WT).

DNL Constructs

Monospecific MAb-IFNα (20-2b-2b, 734-2b-2b and C₂₋₂b-2b) and thebispecific HexAb (20-C2-C2) were generated by combination of anIgG-AD2-module with DDD2-modules using the DNL method, as described inthe preceding Examples. The 734-2b-2b, which comprises tetrameric IFNα2band MAb h734 [anti-Indium-DIVA IgG₁], was used as a non-targetingcontrol MAb-IFNα.

The construction of the mammalian expression vector as well as thesubsequent generation of the production clones and the purification ofC_(H)3-AD2-IgG-v-mab are disclosed in the preceding Examples. Theexpressed recombinant fusion protein has the AD2 peptide linked to thecarboxyl terminus of the C_(H)3 domain of v-mab via a 15 amino acid longflexible linker peptide. Co-expression of the heavy chain-AD2 and lightchain polypeptides results in the formation of an IgG structure equippedwith two AD2 peptides. The expression vector was transfected into Sp/ESFcells (an engineered cell line of Sp2/0) by electroporation. The pdHL2vector contains the gene for dihydrofolate reductase, thus allowingclonal selection, as well as gene amplification with methotrexate (MTX).Stable clones were isolated from 96-well plates selected with mediacontaining 0.2 μM MTX. Clones were screened for C_(H)3-AD2-IgG-vmabproductivity via a sandwich ELISA. The module was produced in rollerbottle culture with serum-free media.

The DDD-module, IFNα2b-DDD2, was generated as discussed above byrecombinant fusion of the DDD2 peptide to the carboxyl terminus of humanIFNα2b via an 18 amino acid long flexible linker peptide. As is the casefor all DDD-modules, the expressed fusion protein spontaneously forms astable homodimer.

The C_(H)1-DDD2-Fab-hL243 expression vector was generated fromhL243-IgG-pdHL2 vector by excising the sequence for theC_(H)1-Hinge-C_(H)2-C_(H)3 domains with SacII and EagI restrictionenzymes and replacing it with a 507 bp sequence encoding C_(H)1-DDD2,which was excised from the C-DDD2-hMN-14-pdHL2 expression vector withthe same enzymes. Following transfection of C_(H)1-DDD2-Fab-hL243-pdHL2into Sp/ESF cells by electroporation, stable, MTX-resistant clones werescreened for productivity via a sandwich ELISA using 96-well microtiterplates coated with mouse anti-human kappa chain to capture the fusionprotein, which was detected with horseradish peroxidase-conjugated goatanti-human Fab. The module was produced in roller bottle culture.

Roller bottle cultures in serum-free H—SFM media and fed-batchbioreactor production resulted in yields comparable to other IgG-AD2modules and cytokine-DDD2 modules generated to date.C_(H)3-AD2-IgG-v-mab and IFNα2b-DDD2 were purified from the culturebroths by affinity chromatography using MABSELECT™ (GE Healthcare) andHIS-SELECT® HF Nickel Affinity Gel (Sigma), respectively, as describedpreviously (Rossi et al., Blood 2009, 114:3864-71). The culture brothcontaining the C_(H)1-DDD2-Fab-hL243 module was applied directly toKAPPASELECT® affinity gel (GE-Healthcare), which was washed to baselinewith PBS and eluted with 0.1 M Glycine, pH 2.5.

The purity of the DNL modules was assessed by SDS-PAGE and SE-HPLC (notshown). Analysis under non-reducing conditions showed that, prior to theDNL reaction, IFNα₂b-DDD2 and C_(H)1-DDD2-Fab-hL243 exist asdisulfide-linked dimers (not shown). This phenomenon, which is alwaysseen with DDD-modules, is beneficial, as it protects the reactivesulfhydryl groups from irreversible oxidation. In comparison,C_(H)3-AD2-IgG-v-mab (not shown) exists as both a monomer and adisulfide-linked dimer, and is reduced to monomer during the DNLreaction. SE-HPLC analyses agreed with the non-reducing SDS-PAGEresults, indicating monomeric species as well as dimeric modules thatwere converted to monomeric forms upon reduction (not shown). Thesulfhydryl groups are protected in both forms by participation indisulfide bonds between AD2 cysteine residues. Reducing SDS-PAGEdemonstrated that each module was purified to near homogeneity andidentified the component polypeptides comprising each module (notshown). For C_(H)3-AD2-IgG-v-mab, heavy chain-AD2 and kappa light chainswere identified. hL243-Fd-DDD2 and kappa light chain polypeptides wereresolved for C_(H)1-DDD2-Fab-hL243 (not shown). One major and one minorband were resolved for IFNα₂b-DDD2 (not shown), which were determined tobe non-glycosylated and O-glycosylated species, respectively.

Generation of 20-C2-2b by DNL

Three DNL modules (C_(H)3-AD2-IgG-v-mab, C_(H)1-DDD2-Fab-hL243, andIFN-α2b-DDD2) were combined in equimolar quantities to generate thebsMAb-IFNα, 20-C2-2b. Following an overnight docking step under mildreducing conditions (1 mM reduced glutathione) at room temperature,oxidized glutathione was added (2 mM) to facilitate disulfide bondformation (locking). The 20-C2-2b was purified to near homogeneity usingthree sequential affinity chromatography steps. Initially, the DNLmixture was purified with Protein A (MABSELECT™), which binds theC_(H)3-AD2-IgG-v-MAb group and eliminates un-reacted IFNα₂b-DDD2 orC_(H)1-DDD2-Fab-hL243. The Protein A-bound material was further purifiedby IMAC using HIS-SELECT® HF Nickel Affinity Gel, which bindsspecifically to the IFNα₂b-DDD2 moiety and eliminates any constructslacking this group. The final process step, using an hL243-anti-idiotypeaffinity gel removed any molecules lacking C_(H)1-DDD2-Fab-hL243.

The skilled artisan will realize that affinity chromatography may beused to purify DNL complexes comprising any combination of effectormoieties, so long as ligands for each of the three effector moieties canbe obtained and attached to the column material. The selected DNLconstruct is the one that binds to each of three columns containing theligand for each of the three effector moieties and can be eluted afterwashing to remove unbound complexes.

The following Example is representative of several similar preparationsof 20-C2-2b. Equimolar amounts of C_(H)3-AD2-IgG-v-mab (15 mg),C_(H)1-DDD2-Fab-hL243 (12 mg), and IFN-α2b-DDD2 (5 mg) were combined in30-mL reaction volume and 1 mM reduced glutathione was added to thesolution. Following 16 h at room temperature, 2 mM oxidized glutathionewas added to the mixture, which was held at room temperature for anadditional 6 h. The reaction mixture was applied to a 5-mL Protein Aaffinity column, which was washed to baseline with PBS and eluted with0.1 M Glycine, pH 2.5. The eluate, which contained ˜20 mg protein, wasneutralized with 3 M Tris-HCl, pH 8.6 and dialyzed into HIS-SELECT®binding buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH₂PO₄, pH 8.0)prior to application to a 5-mL HIS-SELECT® IMAC column. The column waswashed to baseline with binding buffer and eluted with 250 mM imidazole,150 mM NaCl, 50 mM NaH₂PO₄, pH 8.0.

The IMAC eluate, which contained ˜11.5 mg of protein, was applieddirectly to a WP (anti-hL243) affinity column, which was washed tobaseline with PBS and eluted with 0.1 M glycine, pH 2.5. The processresulted in 7 mg of highly purified 20-C2-2b. This was approximately 44%of the theoretical yield of 20-C2-2b, which is 50% of the total startingmaterial (16 mg in this example) with 25% each of 20-2b-2b and 20-C₂-C₂produced as side products.

Generation and CHARACTERIZATION of 20-C2-2b

The bispecific MAb-IFNα was generated by combining the IgG-AD2 module,C_(H)3-AD2-IgG-v-mab, with two different dimeric DDD-modules,C_(H)1-DDD2-Fab-hL243 and IFNα₂b-DDD2. Due to the random association ofeither DDD-module with the two AD2 groups, two side-products, 20-C₂-C₂and 20-2b-2b are expected to form, in addition to 20-C2-2b.

Non-reducing SDS-PAGE (not shown) resolved 20-C2-2b (˜305 kDa) as acluster of bands positioned between those of 20-C2-C2 (˜365 kDa) and20-2b-2b (255 kDa). Reducing SDS-PAGE resolved the five polypeptides(v-mab HC-AD2, hL243 Fd-DDD2, IFNα2b-DDD2 and co-migrating v-mab andhL243 kappa light chains) comprising 20-C2-2b (not shown). IFNα2b-DDD2and hL243 Fd-DDD2 are absent in 20-C₂-C₂ and 20-2b-2b. MABSELECT™ bindsto all three of the major species produced in the DNL reaction, butremoves any excess IFNα2b-DDD2 and C_(H)1-DDD2-Fab-hL243. TheHIS-SELECT® unbound fraction contained mostly 20-C₂-C₂ (not shown). Theunbound fraction from WT affinity chromatography comprised 20-2b-2b (notshown). Each of the samples was subjected to SE-HPLC andimmunoreactivity analyses, which corroborated the results andconclusions of the SDS-PAGE analysis.

Following reduction of 20-C2-2b, its five component polypeptides wereresolved by RP-HPLC and individual ESI-TOF deconvoluted mass spectrawere generated for each peak (not shown). Native, but notbacterially-expressed recombinant IFNα2, is O-glycosylated at Thr-106(Adolf et al., Biochem J 1991; 276 (Pt 2):511-8). We determined that˜15% of the polypeptides comprising the IFNα2b-DDD2 module areO-glycosylated and can be resolved from the non-glycosylatedpolypeptides by RP-HPLC and SDS-PAGE (not shown). LC/MS analysis of20-C2-2b identified both the O-glycosylated and non-glycosylated speciesof IFNα2b-DDD2 with mass accuracies of 15 ppm and 2 ppm, respectively(not shown). The observed mass of the O-glycosylated form indicates anO-linked glycan having the structure NeuGc-NeuGc-Gal-GalNAc, which wasalso predicted (<1 ppm) for 20-2b-2b (not shown). LC/MS identified bothv-mab and hL243 kappa chains as well as hL243-Fd-DDD2 (not shown) assingle, unmodified species, with observed masses matching the calculatedones (<35 ppm). Two major glycoforms of v-mab HC-AD2 were identified ashaving masses of 53,714.73 (70%) and 53,877.33 (30%), indicating G0F andG1F N-glycans, respectively, which are typically associated with IgG(not shown). The analysis also confirmed that the amino terminus of theHC-AD2 is modified to pyroglutamate, as predicted for polypeptideshaving an amino terminal glutamine.

SE-HPLC analysis of 20-C2-2b resolved a predominant protein peak with aretention time (6.7 min) consistent with its calculated mass and betweenthose of the larger 20-C₂-C₂ (6.6 min) and smaller 20-2b-2b (6.85 min),as well as some higher molecular weight peaks that likely representnon-covalent dimers formed via self-association of IFNα2b (not shown).

Immunoreactivity assays demonstrated the homogeneity of 20-C2-2b witheach molecule containing the three functional groups (not shown).Incubation of 20-C2-2b with an excess of antibodies to any of the threeconstituent modules resulted in quantitative formation of high molecularweight immune complexes and the disappearance of the 20-C2-2b peak (notshown). The HIS-SELECT® and WT affinity unbound fractions were notimmunoreactive with WT and anti-IFNα, respectively (not shown). TheMAb-IFNα showed similar binding avidity to their parental MAbs (notshown).

IFNα Biological Activity

The specific activities for various MAb-IFNα were measured using acell-based reporter gene assay and compared to peginterferon alfa-2b(not shown). Expectedly, the specific activity of 20-C2-2b (2454IU/μmol), which has two IFNα2b groups, was significantly lower thanthose of 20-2b-2b (4447 IU/μmol) or 734-2b-2b (3764 IU/μmol), yetgreater than peginterferon alfa-2b (P<0.001) (not shown). The differencebetween 20-2b-2b and 734-2b-2b was not significant. The specificactivity among all agents varies minimally when normalized to IU/μmol oftotal IFNα. Based on these data, the specific activity of each IFNα2bgroup of the MAb-IFNα is approximately 30% of recombinant IFNα2b (4000IU/μmol).

In the ex-vivo setting, the 20-C2-2b DNL construct depleted lymphomacells more effectively than normal B cells and had no effect on T cells(not shown). However, it did efficiently eliminate monocytes (notshown). Where v-mab had no effect on monocytes, depletion was observedfollowing treatment with hL243α4p and MAb-IFNα, with 20-2b-2b and734-2b-2b exhibiting similar toxicity (not shown). Therefore, thepredictably higher potency of 20-C2-2b is attributed to the combinedactions of anti-HLA-DR and IFNα, which may be augmented by HLA-DRtargeting. These data suggest that monocyte depletion may be apharmacodynamic effect associated anti-HLA-DR as well as IFNα therapy;however, this side affect would likely be transient because the monocytepopulation should be repopulated from hematopoietic stem cells.

The skilled artisan will realize that the approach described here toproduce and use bispecific immunocytokine, or other DNL constructscomprising three different effector moieties, may be utilized with anycombinations of antibodies, antibody fragments, cytokines or othereffectors that may be incorporated into a DNL construct, for example thecombination of anti-CD20 and anti-CD22 with IFNα2b.

Example 13 Hexavalent DNL Constructs

The DNL technology described above for formation of trivalent DNLcomplexes was applied to generate hexavalent IgG-based DNL structures(HIDS). Because of the increased number of binding sites for targetantigens, hexavalent constructs might be expected to show greateraffinity and/or efficacy against target cells. Two types of modules,which were produced as recombinant fusion proteins, were combined togenerate a variety of HIDS. Fab-DDD2 modules were as described for usein generating trivalent Fab structures (Rossi et al. Proc Natl Acad SciUSA. 2006; 103(18): 6841-6). The Fab-DDD2 modules form stable homodimersthat bind to AD2-containing modules. To generate HIDS, two types ofIgG-AD2 modules were created to pair with the Fab-DDD2 modules:C-H-AD2-IgG and N-L-AD2-IgG.

C-H-AD2-IgG modules have an AD2 peptide fused to the carboxyl terminus(C) of the heavy (H) chain of IgG via a peptide linker. The DNA codingsequences for the linker peptide followed by the AD2 peptide are coupledto the 3′ end of the CH3 (heavy chain constant domain 3) coding sequenceby standard recombinant DNA methodologies, resulting in a contiguousopen reading frame. When the heavy chain-AD2 polypeptide is co-expressedwith a light chain polypeptide, an IgG molecule is formed possessing twoAD2 peptides, which can therefore bind two Fab-DDD2 dimers. TheC-H-AD2-IgG module can be combined with any Fab-DDD2 module to generatea wide variety of hexavalent structures composed of an Fc fragment andsix Fab fragments. If the C-H-AD2-IgG module and the Fab-DDD2 module arederived from the same parental monoclonal antibody (MAb) the resultingHIDS is monospecific with 6 binding arms to the same antigen. If themodules are instead derived from two different MAbs then the resultingHIDS are bispecific, with two binding arms for the specificity of theC-H-AD2-IgG module and 4 binding arms for the specificity of theFab-DDD2 module.

N-L-AD2-IgG is an alternative type of IgG-AD2 module in which an AD2peptide is fused to the amino terminus (N) of the light (L) chain of IgGvia a peptide linker. The L chain can be either Kappa (K) or Lambda (λ)and will also be represented as K. The DNA coding sequences for the AD2peptide followed by the linker peptide are coupled to the 5′ end of thecoding sequence for the variable domain of the L chain (V_(L)),resulting in a contiguous open reading frame. When the AD2-kappa chainpolypeptide is co-expressed with a heavy chain polypeptide, an IgGmolecule is formed possessing two AD2 peptides, which can therefore bindtwo Fab-DDD2 dimers. The N-L-AD2-IgG module can be combined with anyFab-DDD2 module to generate a wide variety of hexavalent structurescomposed of an Fc fragment and six Fab fragments.

The same technique has been utilized to produce DNL complexes comprisingan IgG moiety attached to four effector moieties, such as cytokines. Inan exemplary embodiment, an IgG moiety was attached to four copies ofinterferon-α2b. The antibody-cytokine DNL construct exhibited superiorpharmacokinetic properties and/or efficacy compared to PEGylated formsof interferon-α2b.

Example 14 Creation of C-H-AD2-IgG-pdHL2 Expression Vectors

The pdHL2 mammalian expression vector has been used to mediate theexpression of many recombinant IgGs. A plasmid shuttle vector wasproduced to facilitate the conversion of any IgG-pdHL2 vector into aC-H-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) wasamplified using the pdHL2 vector as a template and a pair of primers.The amplifier was cloned in the PGEMT® PCR cloning vector. The Fc insertfragment was excised from PGEMT® with XbaI and BamHI restriction enzymesand ligated with AD2-pdHL2 vector that was prepared by digestion ofh679-Fab-AD2-pdHL2 with XbaI and BamHI, to generate the shuttle vectorFc-AD2-pdHL2.

To convert any IgG-pdHL2 expression vector to a C-H-AD2-IgG-pdHL2expression vector, an 861 bp BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 bp BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the CH3domain and NdeI cuts downstream (3′) of the expression cassette.

Example 15 Production of AD2-Linked IgG Species

Production of C-H-AD2-hLL2 IgG

Epratuzumab, or hLL2 IgG, is a humanized anti-human CD22 MAb. Anexpression vector for C-H-AD2-hLL2 IgG was generated from hLL2IgG-pdHL2, as described above, and used to transfect Sp2/0 myeloma cellsby electroporation. Following transfection, the cells were plated in96-well plates and transgenic clones were selected in media containingmethotrexate. Clones were screened for C-H-AD2-hLL2 IgG productivity bya sandwich ELISA using 96-well microtiter plates coated with anhLL2-specific anti-idiotype MAb and detection with peroxidase-conjugatedanti-human IgG. Clones were expanded to roller bottles for proteinproduction and C-H-AD2-hLL2 IgG was purified from the spent culturemedia in a single step using Protein-A affinity chromatography. SE-HPLCanalysis resolved two protein peaks (not shown). The retention time ofthe slower eluted peak was similar to hLL2 IgG (not shown). Theretention time of the faster eluted peak was consistent with a ˜300 kDaprotein (not shown). It was later determined that this peak representsdisulfide linked dimers of C-H-AD2-hLL2-IgG. This dimer is reduced tothe monomeric form during the DNL reaction. SDS-PAGE analysisdemonstrated that the purified C-H-AD2-hLL2-IgG consisted of bothmonomeric and disulfide-linked dimeric forms of the module (not shown).Protein bands representing these two forms are evident by SDS-PAGE undernon-reducing conditions, while under reducing conditions all of theforms are reduced to two bands representing the constituent polypeptides(Heavy chain-AD2 and kappa chain) (not shown). No other contaminatingbands were detected.

Production of C-H-AD2-hA20 IgG

hA20 IgG is a humanized anti-human CD20 MAb. An expression vector forC-H-AD2-hA20 IgG was generated from hA20 IgG-pDHL2, as described above,and used to transfect Sp2/0 myeloma cells by electroporation. Followingtransfection, the cells were plated in 96-well plates and transgenicclones were selected in media containing methotrexate. Clones werescreened for C-H-AD2-hA20 IgG productivity by a sandwich ELISA using96-well microtiter plates coated with a hA20-specific anti-idiotype MAband detection with peroxidase-conjugated anti-human IgG. Clones wereexpanded to roller bottles for protein production and C—H-AD2-hA20 IgGwas purified from the spent culture media in a single step usingProtein-A affinity chromatography. SE-HPLC and SDS-PAGE analyses gavevery similar results to those obtained for C-H-AD2-hLL2 IgG (not shown).

Production of N-L-AD2-hA20 IgG

A 197 bp DNA duplex comprising the coding sequence for the light chainleader peptide, AD2, a 13-residue peptide linker and the first fourresidues of hA20 Vk (all in frame) was generated as follows. Two 100-mersynthetic oligonucleotides, which overlap by 35 base-pairs, were madefully duplex by primer extension using Taq polymerase. The sequence wasamplified by PCR, which appended XbaI and PvuII restriction sites to the5′ and 3′ ends, respectively. The amplifier was cloned into PGEMT®.

The 197 bp XbaI/PvuII fragment was excised from PGEMT® and ligated withthe hA20 V_(K) shuttle vector h2B8-V_(k)-pBR2, which was prepared bydigestion with XbaI and PvuII. The new shuttle vector isAD2-K-hA20-pBR2. A 536 bp XbaI/Bam HI restriction fragment was excisedfrom AD2-K-hA20-pBR2 and ligated with hA20-IgG-pDHL2 vector that wasprepared by digestion with XbaI and Bam HI to generate the expressionvector N-L-AD2-hA20-IgG-pdHL2.

N-L-AD2-hA20-IgG-pdHL2 was used to transfect Sp2/0 myeloma cells byelectroporation. Following transfection, the cells were plated in96-well plates and transgenic clones were selected in media containingmethotrexate. Clones were screened for N-L-AD2-hA20 IgG productivity bya sandwich ELISA using 96-well microtiter plates coated with ahA20-specific anti-idiotype MAb and detection with peroxidase-conjugatedanti-human IgG. Clones were expanded to roller bottles for proteinproduction and N-L-AD2-hA20 IgG was purified from the spent culturemedia in a single step using Protein-A affinity chromatography.

Size exclusion HPLC showed that the majority of the N-L-AD2-hA20 IgG inthe prep is in a monomeric form with a retention time similar to IgG(not shown). Two additional peaks likely representing disulfide linkeddimeric and trimeric forms and each accounting for approximately 15% ofthe total protein were also observed (not shown). Mild reduction of theprep, as is used in the DNL reaction, resulted in the conversion of thedimeric and trimeric forms to the monomeric form (not shown).

Example 16 Generation of Hexavalent DNL Constructs

Generation of Hex-hA20

The DNL method was used to create Hex-hA20, a monospecific anti-CD20RIDS, by combining C-H-AD2-hA20 IgG with hA20-Fab-DDD2. The Hex-hA20structure contains six anti-CD20 Fab fragments and an Fc fragment,arranged as four Fab fragments and one IgG antibody. Hex-hA20 was madein four steps.

Step 1, Combination: A 210% molar equivalent of (hA20-Fab-DDD2)₂ wasmixed with C-H-AD2-hA20 IgG. This molar ratio was used because twoFab-DDD2 dimers are coupled to each C-H-AD2-hA20 IgG molecule and anadditional 10% excess of the former ensures that the coupling reactionis complete. The molecular weights of C-H-AD2-hA20 IgG and(hA20-Fab-DDD2)₂ are 168 kDa and 107 kDa, respectively. As an example,134 mg of hA20-Fab-DDD2 would be mixed with 100 mg of C-H-AD2-hA20 IgGto achieve a 210% molar equivalent of the former. The mixture istypically made in phosphate buffered saline, pH 7.4 (PBS) with 1 mMEDTA.

Step 2, Mild Reduction: Reduced glutathione (GSH) was added to a finalconcentration of 1 mM and the solution is held at room temperature(16-25° C.) for 1-24 hours.

Step 3, Mild Oxidation: Following reduction, oxidized glutathione (GSSH)was added directly to the reaction mixture to a final concentration of 2mM and the solution was held at room temperature for 1-24 hours.

Step 4, Isolation of the DNL product: Following oxidation, the reactionmixture was loaded directly onto a Protein-A affinity chromatographycolumn. The column was washed with PBS and the Hex-hA20 was eluted with0.1 M glycine, pH 2.5. Since excess hA20-Fab-DDD2 was used in thereaction, there was no unconjugated C-H-AD2-hA20 IgG, or incomplete DNLstructures containing only one (hA20-Fab-DDD2)₂ moiety. The unconjugatedexcess hA20-Fab-DDD2 does not bind to the affinity resin. Therefore, theProtein A-purified material contains only the desired product.

The calculated molecular weight from the deduced amino acid sequences ofthe constituent polypeptides is 386 kDa. Size exclusion HPLC analysisshowed a single protein peak with a retention time consistent with aprotein structure of 375-400 kDa (not shown). SDS-PAGE analysis undernon-reducing conditions showed a cluster of high molecular weight bandsindicating a large covalent structure (not shown). SDS-PAGE underreducing conditions showed the presence of only the three expectedpolypeptide chains: the AD2-fused heavy chain (HC-AD2), the DDD2-fusedFd chain (Fd-DDD2), and the kappa chains (not shown).

Generation of Hex-hLL2

The DNL method was used to create a monospecific anti-CD22 HIDS(Hex-hLL2) by combining C-H-AD2-hLL2 IgG with hLL2-Fab-DDD2. The DNLreaction was accomplished as described above for Hex-hA20. Thecalculated molecular weight from the deduced amino acid sequences of theconstituent polypeptides is 386 kDa. Size exclusion HPLC analysis showeda single protein peak with a retention time consistent with a proteinstructure of 375-400 kDa (not shown). SDS-PAGE analysis undernon-reducing conditions showed a cluster of high molecular weight bands,which were eliminated under reducing conditions to leave only the threeexpected polypeptide chains: HC-AD2, Fd-DDD2, and the kappa chain (notshown).

Generation of DNL1 and DNL1C

The DNL method was used to create bispecific HIDS by combiningC-H-AD2-hLL2 IgG with either hA20-Fab-DDD2 to obtain DNL1 or hMN-14-DDD2to obtain DNL1C. DNL1 has four binding arms for CD20 and two for CD22.As hMN-14 is a humanized MAb to carcinoembryonic antigen (CEACAM5),DNL1C has four binding arms for CEACAM5 and two for CD22. The DNLreactions were accomplished as described for Hex-hA20 above.

For both DNL1 and DNL1C, the calculated molecular weights from thededuced amino acid sequences of the constituent polypeptides are ˜386kDa. Size exclusion HPLC analysis showed a single protein peak with aretention time consistent with a protein structure of 375-400 kDa foreach structure (not shown). SDS-PAGE analysis under non-reducingconditions showed a cluster of high molecular weight bands, which wereeliminated under reducing conditions to leave only the three expectedpolypeptides: HC-AD2, Fd-DDD2, and the kappa chain (not shown).

Generation of DNL2 and DNL2C

The DNL method was used to create bispecific HIDS by combiningC-H-AD2-hA20 IgG with either hLL2-Fab-DDD2 to obtain DNL2 or hMN-14-DDD2to obtain DNL2C. DNL2 has four binding arms for CD22 and two for CD20.DNL2C has four binding arms for CEACAM5 and two for CD20. The DNLreactions were accomplished as described for Hex-hA20.

For both DNL2 and DNL2C, the calculated molecular weights from thededuced amino acid sequences of the constituent polypeptides are ˜386kDa. Size exclusion HPLC analysis showed a single protein peak with aretention time consistent with a protein structure of 375-400 kDa foreach structure (not shown). SDS-PAGE analysis under non-reducingconditions showed high molecular weight bands, but under reducingconditions consisted solely of the three expected polypeptides: HC-AD2,Fd-DDD2, and the kappa chain (not shown).

Generation of K-Hex-hA20

The DNL method was used to create a monospecific anti-CD20 HIDS(K-Hex-hA20) by combining N-L-AD2-hA20 IgG with hA20-Fab-DDD2. The DNLreaction was accomplished as described above for Hex-hA20.

The calculated molecular weight from the deduced amino acid sequences ofthe constituent polypeptides is 386 kDa. SDS-PAGE analysis undernon-reducing conditions showed a cluster of high molecular weight bands,which under reducing conditions were composed solely of the fourexpected polypeptides: Fd-DDD2, H-chain, kappa chain, and AD2-kappa (notshown).

Generation of DNL3

A bispecific HIDS was generated by combining N-L-AD2-hA20 IgG withhLL2-Fab-DDD2. The DNL reaction was accomplished as described above forHex-hA20. The calculated molecular weight from the deduced amino acidsequences of the constituent polypeptides is 386 kDa. Size exclusionHPLC analysis showed a single protein peak with a retention timeconsistent with a protein structure of 375-400 kDa (not shown). SDS-PAGEanalysis under non-reducing conditions showed a cluster of highmolecular weight bands that under reducing conditions showed only thefour expected polypeptides: Fd-DDD2, H-chain, kappa chain, and AD2-kappa(not shown).

Stability in Serum

The stability of DNL1 and DNL2 in human serum was determined using abispecific ELISA assay. The protein structures were incubated at 10μg/ml in fresh pooled human sera at 37° C. and 5% CO₂ for five days. Forday 0 samples, aliquots were frozen in liquid nitrogen immediately afterdilution in serum. ELISA plates were coated with an anti-Id to hA20 IgGand bispecific binding was detected with an anti-Id to hLL2 IgG. BothDNL1 and DNL2 were highly stable in serum and maintained completebispecific binding activity (not shown).

Binding Activity

The HIDS generated as described above retained the binding properties oftheir parental Fab/IgGs. Competitive ELISAs were used to investigate thebinding avidities of the various HIDS using either a rat anti-idiotypeMAb to hA20 (WR2) to assess the binding activity of the hA20 componentsor a rat anti-idiotype MAb to hLL2 (WN) to assess the binding activityof the hLL2 components. To assess hA20 binding, ELISA plates were coatedwith hA20 IgG and the HIDS were allowed to compete with the immobilizedIgG for WR2 binding. To assess hLL2 binding, plates were coated withhLL2 IgG and the HIDS were allowed to compete with the immobilized IgGfor WN binding. The relative amount of anti-Id bound to the immobilizedIgG was detected using peroxidase-conjugated anti-Rat IgG.

Examining the relative CD20 binding avidities (FIG. 1A), DNL2, which hastwo CD20 binding groups, showed a similar binding avidity to hA20 IgG,which also has two CD20-binding arms (FIG. 1A). DNL1, which has fourCD20-binding groups, had a stronger (˜4-fold) relative avidity than DNL2or hA20 IgG (FIG. 1A). Hex-hA20, which has six CD20-binding groups, hadan even stronger (˜10-fold) relative avidity than hA20 IgG (FIG. 1A).

Similar results were observed for CD22 binding (FIG. 1B). DNL1, whichhas two CD20 binding groups, showed a similar binding avidity to hLL2IgG, which also has two CD22-binding arms (FIG. 1B). DNL2, which hasfour CD22-binding groups, had a stronger (>5-fold) relative avidity thanDNL1 or hLL2 IgG. Hex-hLL2, which has six CD22-binding groups, had aneven stronger (>10-fold) relative avidity than hLL2 IgG (FIG. 1B).

As both DNL2 and DNL3 contain two hA20 Fabs and four hLL2 Fabs, theyshowed similar strength in binding to the same anti-id antibody (notshown).

Some of the HIDS were observed to have potent anti-proliferativeactivity on lymphoma cell lines. DNL1, DNL2 and Hex-hA20 inhibited cellgrowth of Daudi Burkitt Lymphoma cells in vitro (FIG. 2). Treatment ofthe cells with 10 nM concentrations was substantially more effective forthe HIDS compared to rituximab (not shown). Using a cell counting assay,the potency of DNL1 and DNL2 was estimated to be more than 100-foldgreater than that of rituximab, while the Hex-hA20 was shown to be evenmore potent (not shown). This was confirmed with an MTS proliferationassay in which dose-response curves were generated for Daudi cellstreated with a range of concentrations of the HIDS (not shown). Comparedto rituximab, the bispecific HIDS (DNL1 and DNL2) and Hex-hA20were >100-fold and >10000-fold more potent, respectively.

Example 17 In Vivo Anti-Tumor Activity of Hexavalent DNL Constructs

The HIDS were shown to have therapeutic efficacy in vivo using a humanBurkitt Lymphoma model in mice (FIG. 3). Low doses (12 μg) of DNL2 andHex-hA20 more than doubled the survival times of tumor bearing mice.Treatment with higher doses (60 μg) resulted in long-term survivors.

Example 18 Comparative Effects of Hexavalent DNL Constructs and ParentIgG on Lymphoma Cell Lines

Dose-response curves for HIDS (DNL1, DNL2, Hex-hA20) versus a parent IgG(hA20 IgG) were compared for three different lymphoma cell lines (FIG.4), using an MTS proliferation assay. In Daudi lymphoma cells (FIG. 4,top panel), the bispecific structures DNL1 (not shown) and DNL2showed >100-fold more potent anti-proliferative activity and Hex-hA20showed >10,000-fold more potent activity than the parent hA20 IgG.Hex-hLL2 and the control structures (DNL1-C and DNL2-C) had very littleanti-proliferative activity in this assay (not shown).

In Raji lymphoma cells (FIG. 4, middle panel), Hex-hA20 displayed potentanti-proliferative activity, but DNL2 showed only minimal activitycompared with hA20 IgG. In Ramos lymphoma cells (FIG. 4, bottom panel),both DNL2 and Hex-hA20 displayed potent anti-proliferative activity,compared with hA20 IgG. These results show that the increased potency ofBIDS relative to the parent IgGs is not limited to particular celllines, but rather is a general phenomenon for cells displaying theappropriate targets.

Example 19 CDC and ADCC Activity of Hexavalent DNL Constructs

In vivo, anti-CD20 monoclonal antibodies such as rituximab and hA20 canutilize complement-dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC) and signal transduction induced growthinhibition/apoptosis for tumor cell killing. The hexavalent DNLstructures (DNL1, DNL2, Hex-hA20) were tested for CDC activity usingDaudi cells in an in vitro assay. Surprisingly, none of the hexavalentstructures that bind CD20 exhibited CDC activity (not shown). The parenthA20 IgG exhibited potent CDC activity (not shown), while as expectedthe hLL2 antibody against CD22 showed no activity (not shown). The lackof effect of DNL2 and Hex-hA20 was of interest, since they comprisehA20-IgG-Ad2, which showed similar positive CDC activity to hA20 IgG(not shown).

DNL1 was assayed for ADCC activity using freshly isolated peripheralblood mononuclear cells. Both rituximab and hA20 IgG showed potentactivity on Daudi cells, while DNL1 did not exhibit any detectable ADCCactivity (not shown).

These data suggest that the Fc region may become inaccessible foreffector functions (CDC and ADCC) when four additional Fab groups aretethered to its carboxyl termini. Therefore, the hexavalent DNLstructures appear to rely only on signal transduction induced growthinhibition/apoptosis for in vivo anti-tumor activity.

Example 20 Multiple Signaling Pathways Induced by Hexavalent,Monospecific Anti-CD20 and Bispecific Anti-CD20/CD22 AntibodiesCorrelate with Enhanced Toxicity to B-Cell Lymphomas

We have generated hexavalent antibodies (HexAbs) comprising 6 Fabstethered to one Fc of human IgG1. Three such constructs, 20-20, amonospecific HexAb comprising 6 Fabs of veltuzumab (humanized anti-CD20immunoglobulin G1κ [IgG1κ]), 20-22, a bispecific HexAb comprisingveltuzumab and 4 Fabs of epratuzumab (humanized anti-CD22 IgG1κ), and22-20, a bispecific HexAb comprising epratuzumab and 4 Fabs ofveltuzumab, were shown to inhibit proliferation of several lymphoma celllines at nanomolar concentrations in the absence of a crosslinkingantibody, as described in the Examples above. We report here a detailedanalysis of the apoptotic and survival signals induced by the 3 HexAbsin Burkitt lymphomas and provide in vitro cytotoxicity data foradditional lymphoma cell lines and also chronic lymphocytic leukemiapatient specimens. Among the key findings are the significant increasein the levels of phosphorylated p38 and PTEN (phosphatase and tensinhomolog deleted on chromosome 10) by all 3 HexAbs and notabledifferences in the signaling events triggered by the HexAbs from thoseincurred by crosslinking veltuzumab or rituximab with a secondaryantibody. Thus, the greatly enhanced direct toxicity of these HexAbscorrelates with their ability to alter the basal expression of variousintracellular proteins involved in regulating cell growth, survival, andapoptosis, with the net outcome leading to cell death.

The goal of this study was to extend our in vitro characterization of20-20, 20-22, and 22-20, with a primary interest in elucidating theintracellular signaling pathways involved in transducing CD20 uponligating human lymphoma cells with each of these 3 HexAbs, and comparingthe results with those obtained in parallel with epratuzumab,veltuzumab, and rituximab. Selective experiments were performed todetermine whether the individual profile of the kinases activated by theanti-CD20/CD22 HexAbs would be similar to or different from thattriggered by anti-IgM, or by crosslinking veltuzumab or rituximab with asecondary antibody. The data presented below allow us to correlate theenhanced direct cytotoxicity of the anti-CD20/CD22 HexAbs, compared withtheir bivalent parental antibodies, with their increased ability toup-regulate PTEN, phosphorylated p38 and cyclin-dependent kinase (CDK)inhibitors, as represented by p21, p27 and Kip2. The reasons forselecting 22-20 as the best lead among the 3 HexAbs for clinicalevaluation also are discussed.

Methods

Antibodies and reagents—The generation and preparation of 20-20, 20-22,and 22-20 was described above. Rituximab was obtained from commercialsupplies. Mouse antihuman IgM was purchased from Southern Biotech. Otherantibodies were from Cell Signaling or Santa Cruz Biotechnology.Horseradish peroxidase-conjugated secondary antibodies were obtainedfrom Jackson ImmunoResearch Laboratories. Heat-inactivated fetal bovineserum was purchased from Hyclone. Cell culture media, supplements,tetramethylrhodamine ethyl ester, and the transfection reagent DMRIE-Cwere from Invitrogen Life Technologies. One Solution MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assay reagent was obtained from Promega. Reducing 4%-20% gradientTris-glycine gels were from Cambrex Bio Science. Annexin V-ALEXA FLUOR®488 conjugate for apoptosis detection was obtained from Invitrogen. Allother chemicals were purchased from Sigma-Aldrich.

Cell culture—Burkitt (Daudi, Raji) and non-Burkitt (RL and DoHH2) humanlymphoma lines were obtained from ATCC and cultured at 37° C. in 5% CO₂and RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovineserum, 2 mM L-glutamine, 200 U/mL penicillin, and 100 μg/mLstreptomycin. Cells from chronic lymphocytic leukemia (CLL) patientswere collected from whole blood by FICOLL-HYPAQUE® separation (Stein etal., Blood 2010, 115:5180-5190) and grown in RPMI media as describedabove.

Cell viability assay—Cells were seeded at a density of 1×10⁵ cells/mL in96-well plates (1×10⁴ cells/well) and incubated with each test antibodyor DNL complex at a final concentration of 0.01-500 nM for 3 (Daudi) and4 days (Raji, R L, and DoHH2). Where indicated, antibodies werecross-linked with a secondary goat anti-human (GAH) antibody at aconcentration of 10 μg/mL. Patient CLL cells were seeded in 48-wellplates at a density of 5×10⁵ cells/mL (1.5×10⁵ cells/well) and incubatedwith each test antibody at a final concentration of 10 nM for 3 days.The number of living cells was then determined using the solubletetrazolium salt MTS following the manufacturer's protocol.

Annexin V binding assay—Cells in 6-well plates (2×10⁵ cells/well at1×10⁵ cells/mL) were treated with each test antibody at 10 or 100 nM for24 hours, washed, resuspended in 100 μL of annexin-binding buffer (10 mMHEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 140 mMNaCl, and 2.5 mM CaCl₂ in phosphate-buffered saline; PBS), stained with5 pt of annexin V-ALEXA FLUOR® 488 conjugate for 20 minutes, followed bystaining with 1 μg/mL of propidium iodide (PI) in 400 μL, ofannexin-binding buffer, and analyzed by flow cytometry (FACSCALIBUR®;Becton Dickinson). Cells stained positive with annexin V (including bothPI-negative and -positive) were counted as apoptotic populations.

Cell-cycle analysis—Cells were seeded and treated with each testantibody as described for the annexin V binding assay, except that theywere resuspended in 0.5 mL of a solution containing PI (50 μg/mL),sodium citrate (0.1%), and Triton X-100 (0.1%) and stained for 1 hourbefore analysis by flow cytometry.

Assessment of mitochondrial membrane depolarization—Changes inmitochondrial transmembrane potential (Δψ_(m)) were determined by flowcytometry. Briefly, Daudi cells in 6-well plates (2×10⁵ cells/wellseeded at 1×10⁵ cells/mL) were incubated overnight (16 hours) withrituximab (133 nM), veltuzumab (133 nM), or each of the 3 HexAbs (100nM). Samples also included rituximab (133 nM) or veltuzumab (133 nM) inthe presence of a crosslinking antibody (10 μg/mL). Cells were stainedfor 30 minutes in the dark at 37° C. with 50 nM of the fluorescent probetetramethylrhodamine ethyl ester in a medium containing antibiotics andserum, washed 3× with PBS, and analyzed.

Immunoblot analysis—In general, Daudi or Raji cells (1×10⁶ cells/mL)were treated for a predetermined time with rituximab, veltuzumab, orepratuzumab at 133 or 10 nM, or with 20-20, 20-22, or 22-20 at 10 nM,then washed in PBS, centrifuged, and lysed in ice cold 1×RIPA buffercomprising 2 mM sodium orthovanadate, 5 mM sodium fluoride, 2.5 mMsodium pyrophosphate, 1 mM β-glycerophosphate, and 0.25% sodiumdeoxycholate. The lysates were centrifuged at 13,000×g, and thesupernatants were collected. Protein content was determined using theprotein assay kit from Bio-Rad, with bovine serum albumin as thestandard. Protein samples (25 μg) were mixed with the lysis buffer andheated at 95° C. for 5 minutes, followed by separation on 4%-20%gradient Tris-glycine gels. Separated proteins were transferredelectrophoretically onto nitrocellulose membranes. Nonspecific bindingsites were blocked in 10 mM Tris-buffered saline containing 0.05%Tween-20 (TBS-T) and 10% nonfat milk. Membranes were incubated with aprimary antibody (1:1000 dilution in TBS-T containing 5% bovine serumalbumin) overnight at 4° C. The next day, membranes were washed 3× withTBS-T at room temperature. Horseradish peroxidase-conjugated secondaryantibody (1:5000 dilution) was used to probe the primary antibody. Blotswere visualized with enhanced chemiluminescence (Thermo Scientific).

RNA interference of PTEN—Daudi cells were transfected with PTEN smallinterfering RNA (siRNA; Cell Signaling Technologies) or control siRNA(Santa Cruz Biotechnology) in E-well plates using DMRIE-C, permanufacturer's instructions, then treated with each HexAb at 10 nM for16 hours. Cells were washed with PBS, stained with annexin V conjugatefor 20 minutes, and analyzed by flow cytometry. Controls also includedcells incubated with only DMRIE-C. Immunoblot analysis was performed asdescribed above with 5 μg protein samples and anti-PTEN antibody.

Results

Growth inhibition and apoptosis—The dose-response curves (FIG. 5A)obtained with the MTS assay from a 3-day treatment of Daudi cellsindicated comparable values of EC₅₀ for 20-20, 22-20, and 20-22 (2.3-5.8nM), which were 100-fold more potent than veltuzumab or rituximab (>500nM). Under these conditions, crosslinking veltuzumab or rituximab withGAH antibody potently inhibited the proliferation of Daudi cells, withan EC₅₀ of approximately 0.6 nM. Previous results (Rossi et al., Blood2009, 113:6161-6171) obtained from a cell counting assay at day 5 inDaudi demonstrated lower EC₅₀ values for 22-20 (0.32 nM) and 20-22 (0.5nM), which may have been due to a longer incubation time (5 vs 3 days),as well as using a different assay (cell counting vs MTS). The 3 HexAbsalso inhibited the proliferation of Raji (FIG. 5B), with EC₅₀ values of6-8 nM, and when tested at 10 nM, were effective in suppressing thegrowth of RL and DoHH2 (FIG. 5C), as well as CLL specimens (Table 7)from 3 of the 8 patients (CLL078, CLL113, and CLL145) showing higherlevels of CD20 expression (Stein et al., Blood 2010, 115:5180-5190). Itis noted that under the same conditions and even in the presence of GAH,neither rituximab nor veltuzumab showed significant inhibition (10% orless) of CLL samples from all 8 patients.

TABLE 7 Cytotoxicity of HexAbs on CLL patient specimens as determined bythe MTS assay^(#) CD20 Rituximab + Veltuzumab + Patient ID expressionRituximab GAB Veltuzumab GAH 20-20 22-20 20-22 CLL021 Low 114 ± 6 120 ±6  113 ± 12  103 ± 11  117 ± 2  118 ± 6  114 ± 4  CLL022 Low 102 ± 5 107± 5  106 ± 1  102 ± 3  114 ± 1  113 ± 1  99 ± 2  CLL030 Low 107 ± 2 105± 2  105 ± 3  105 ± 16  108 ± 3  115 ± 2  104 ± 3  CLL037 Low 107 ± 5102 ± 18  102 ± 8  90 ± 4  109 ± 4  99 ± 3  96 ± 6  CLL078 Moderate 104± 1 98 ± 2  101 ± 3  93 ± 4  74 ± 5  76 ± 1  72 ± 2  CLL113 High 101 ± 197 ± 4  92 ± 1  96 ± 3  45 ± 2  43 ± 3  39 ± 5  CLL117 Low 107 ± 9 98 ±7  98 ± 8  97 ± 1  101 ± 7  102 ± 5  84 ± 2  CLL145 Moderate 100 ± 4 107± 3  106 ± 6  101 ± 9   84 ± 1  79 ± 1  73 ± 2  ^(#)Values shown arepercentage of untreated controls. Cells were treated with each antibodyat 10 nM and, where indicated, GAH at 10 μg/mL. MTS indicates theviability assay using One Solution assay reagent; and GAH, goatanti-human secondary antibody used for cross linking.

We next determined whether the observed growth inhibition would involveapoptosis. As shown in FIG. 5D, treating Daudi cells for 24 hours withthe 3 HexAbs resulted in approximately 20%-30% apoptosis at 10 nM, andapproximately 25%-35% at 100 nM. In contrast, the parental antibodies(veltuzumab and epratuzumab) and also rituximab, at 10 nM, andepratuzumab at 100 nM, did not induce apoptosis beyond the backgroundlevels observed with the untreated control. However, a slight increasewas observed for the 2 anti-CD20 antibodies, veltuzumab and rituximab,at 100 nM. These results confirm the findings above that 20-20, 22-20,and 20-22 are effective in inducing apoptosis without the requirement ofa crosslinking antibody, and suggest that apoptosis of Daudi cells maybe provoked with higher concentrations of a Type I anti-CD20 antibody(Cragg & Glennie, Blood 2004, 103:2738-2743), as represented byveltuzumab or rituximab. Similar results were obtained with Raji cells,in which the 3 HexAbs induced approximately 20%-30% apoptosis whentested at 10 or 100 nM (data not shown).

Differentiation from anti-IgM and cross-linked anti-CD20—Ligation of theB-cell antigen receptor (BCR) with anti-IgM antibody, or CD20 withveltuzumab or rituximab in the presence of a crosslinking antibody,results in a rapid rise of intracellular calcium (Walshe et al., J BiolChem 2008, 283:16971-1698). Thus, the inability of the 3 anti-CD20/CD22HexAbs to induce a significant increase in calcium flux (Rossi et al.,Blood 2009, 113:6161-6171; Rossi et al., Cancer Res 2008, 68:8384-8392)suggests the involvement of different signals. Noting that activation ofBCR in B cells induces the phosphorylation of Lyn, Syk, and PLCγ2 (Niiro& Clark, Nat Rev Immunol 2002, 2:945-956), we first compared thephosphorylation profiles of these key signaling molecules in Daudi cellstreated for 24 hours with the following: 133 nM of epratuzumab,veltuzumab, or rituximab; 10 nM of 20-20, 22-20, or 20-22; or 10 μg/mLof anti-IgM. As shown in FIG. 6A, anti-IgM induced the phosphorylationof Lyn, Syk, and PLCγ2 significantly above the basal levels observed forthe untreated cells. In contrast, the 3 HexAbs at 10 nM neither inducedthe phosphorylation of Syk nor increased the constitutive level ofphosphorylated PLCγ2. However, they effectively reduced the constitutivelevel of phosphorylated Lyn, which was notable within 2 hours, becamemore prominent with time, and persisted for at least 24 hours (FIG. 6B).Additional studies revealed that the HexAbs diminished the level ofphosphorylated Akt (FIG. 6C) and stimulated the expression of Raf-1kinase inhibition protein (RKIP), which was unchanged with anti-IgM(FIG. 6D). These characteristic changes were also observed forveltuzumab or rituximab at 133 nM (FIG. 6A-C), but not at 10 nM, asshown for phosphorylated Lyn and Akt (FIG. 6E). No appreciable changeswere observed with epratuzumab at 10 or 133 nM.

Modulation of the MAPK pathways—The up-regulation of RKIP prompted us toinvestigate whether these anti-CD20/CD22 HexAbs modulatemitogen-activated protein kinase (MAPK) pathways. Intracellularsignaling by rituximab has been studied intensively and been reported toinfluence various signaling pathways in B-cell malignancies (Bonavida,Oncogene 2007, 26:3629-3636; Jazirehi et al., Mol Cancer Ther 2003,2:1183-1193; Jazirehi et al., Cancer Res 2007, 67:1270-1281; Vega etal., Oncogene 2004, 23:3530-40), in particular, the down-regulation ofboth phosphorylated ERK (extracellular signal regulated kinase)(Jazirehi et al., Cancer Res 2004, 64:7117-7126) and p38 MAPK (Vega etal., Oncogene 2004, 23:3530-40). Daudi cells were treated for 24 hourswith 10 nM of 20-20, 20-22, or 22-20, or 133 nM of epratuzumab,veltuzumab, or rituximab, and whole-cell lysates were subjected toimmunoblotting using phospho-specific ERK and p38 antibodies, with totalERK, p38, and β-actin serving as controls for comparing the amount ofproteins loaded in each sample. As shown in FIG. 7A, both hexavalentmonospecific 20-20 and hexavalent bispecific 20-22 and 22-20, whentested at 10 nM, induced more than a 50% decrease in the levels ofphosphorylated ERK, which was also observed with veltuzumab or rituximabat 133 nM. In addition, all 3 HexAbs led to a 3-fold increase in thelevels of phosphorylated p38, whereas veltuzumab and rituximab appearedto have an opposite or minimal effect on the phosphorylation of p38.Similar results (ie, decrease in phosphorylated ERK and increase inphosphorylated p38) also were observed in Raji cells under the sameconditions (data not shown). The time course study in Daudi revealed acontinuing decrease of phosphorylated ERK during a 24-hour period, whichbegan to be noticeable at 2 hours after the addition of the HexAbs (FIG.7B). In contrast, cross-linking veltuzumab or rituximab with GAHresulted in enhanced phosphorylation of both ERK and p38 (FIG. 7C), thusfurther confirming that the HexAbs act through different mechanisms fromhyper cross-linked anti-CD20 antibodies.

NF-κB pathway, Bcl-2 family proteins, and mitochondrial membranedepolarization—Consistent with the observation of increased RKIP anddecreased phosphorylation of ERK, which should negatively affect thenuclear factor (NF)-κB pathway, we found a significant reduction in thephosphorylation of IKKα/β and IκBα (FIG. 8A). To account for theenhanced potency of the HexAbs to induce apoptosis, we also probed theexpression levels of certain pro- and anti-apoptotic proteins of theBcl-2 family, and the results (FIG. 8B) convincingly demonstrate thedown-regulation of at least 4 anti-apoptotic proteins (Mcl-1, Bcl-xL,Bcl-2, and phospho-BAD), with concurrent up-regulation of onepro-apoptotic protein (Bax) that was evident for 22-20 and 20-22. It isnoted that the reduction in the anti-apoptotic phospho-BAD was not dueto a parallel decrease in the apoptotic BAD, which remained unchanged.Such alterations in the balance of anti- and pro-apoptotic proteins canchange the cell fate from survival to apoptosis. The active modulationof Bcl-2 family proteins further led us to determine whethermitochondrial membrane polarization was involved. Surprisingly, onlycross-linked veltuzumab or rituximab, but not the HexAbs, could inducean appreciable loss of mitochondrial membrane potential in Daudi cellsover the untreated control (FIG. 8C), although they were all capable ofinducing apoptosis under the conditions examined. These results agreewith the notion that the HexAbs differ in mechanisms of action fromcross-linked veltuzumab or rituximab.

Role of PTEN—The up-regulation of RKIP as well as the down-regulation ofthe AKT and NF-κB pathways also prompted us to investigate whether thetumor suppressor, PTEN, plays a specific role in the apoptosis inducedby the HexAbs. Daudi cells were treated with 10 nM 20-20, 20-22, or22-20, or with 133 nM of rituximab, and the cellular levels of PTEN wereexamined at 1, 2, 4, 6, and 24 hours (FIG. 9A). Whereas all 3 HexAbsinduced a notable increase in PTEN at 1 hour, which persisted throughthe next 3-5 hours and returned to the basal level at 24 hours, noappreciable change in PTEN was observed with rituximab during the sameperiod. Because up-regulation of PTEN may result in enhanced apoptosis,down-regulation of PTEN by specific siRNAs (FIG. 9B) should preventapoptosis, as shown in FIG. 9C. The involvement of PTEN was likewiseindicated by the observation that LY294002, the specific inhibitor forPI3K, increased the apoptosis induced by the HexAbs from 20% to 25%-40%,but had no effect on either veltuzumab or rituximab (FIG. 9D).

Deregulation of cell cycle—The HexAbs were found to arrest Daudi cellsin the G₁ phase at both 100 nM (FIG. 10A) and 10 nM (FIG. 10B). TreatingDaudi cells with veltuzumab or rituximab exhibited a similardistribution of various phases as the untreated cells. The deregulationof cell cycle by the HexAbs was associated with the up-regulation of theCDK inhibitors, as shown for p21, p27(Kip1), and Kip2 in FIG. 10C, aswell as the down-regulation of cyclin D1 and phosphorylated Rb (FIG.10D).

Discussion

The HexAbs (20-20, 20-22, and 22-20) share properties of both Type I(rituximab, veltuzumab) and Type II (B1, tositumomab) antibodies (Cragg& Glennie, Blood 2004, 103:2738-2743). Consistent with Type II, theHexAbs are negative for CDC and calcium mobilization, do not requirecrosslinking for growth inhibition or apoptosis, and induce stronghomotypic adhesion; yet, they induce translocation of CD20 to lipidrafts like Type I antibodies (Rossi et al., Blood 2009, 113:6161-6171;Rossi et al., Cancer Res 2008, 68:8384-8392). Unexpectedly, 20-20inhibited proliferation of Burkitt NHL cell lines, Daudi and Raji, invitro with considerably greater potency, compared with either Type I orII antibodies (Rossi et al., Cancer Res 2008, 68:8384-8392). Burkittlymphoma lines, Daudi and Raji, were sensitive to all 3 HexAbs, withsimilar EC₅₀ values (Rossi et al., Blood 2009, 113:6161-6171; Rossi etal., Cancer Res 2008, 68:8384-8392) to cross-linked veltuzumab orrituximab, whereas non-Burkitt lymphoma lines, RL and DoHH2, depictedapproximately 25%-40% inhibition on treatment with 10 nM HexAbs. Thedecrease in potency could have been due to the formation of visibleclumps by RL and DoHH2 cells in suspension.

Direct toxicity of these HexAbs was also evaluated on 8 CLL patientspecimens, which varied in their CD20 expression. The 3 specimensexpressing moderate to high CD20 showed 30%-60% inhibition by theHexAbs, whereas no significant inhibition was observed in the other 5specimens with low CD20 expression. Interestingly, neither rituximab norveltuzumab, with or without crosslinking, produced measurableinhibition. Although these studies suggest a trend in the activity ofHexAbs related to CD20 expression, more patient samples are needed tosubstantiate this. On the other hand, it is intriguing that thesemultivalent monospecific/bispecific HexAbs depict better anti-lymphomaand anti-leukemia properties than their parental IgGs.

To better elucidate the mechanisms by which direct cell killing isachieved with these hexavalent constructs, we focused here on theinvestigation of the signaling pathways that are triggered in Daudicells by the 3 HexAbs, in comparison to those induced by the parentalantibodies, with some of the experiments repeated in Raji cells. We alsoperformed selective studies in which human lymphoma cells were treatedwith anti-IgM antibody to activate the BCR, or with veltuzumab orrituximab in the presence of a cross-linking antibody to enhance theapoptotic potency. Our key findings are summarized as follows: (1) Thesignaling events triggered by 20-20, 22-20, or 20-22 are quantitativelyand qualitatively similar in Daudi cells, but distinct from thoseinduced by anti-IgM. (2) Although veltuzumab and rituximab modify thesignaling events in Daudi cells similarly to the hexavalent derivatives,as observed for the ERK and NF-κB pathways, both require a higherconcentration to be effective and are less efficient in modulating thecell-cycle regulators that promote growth arrest. In addition, thebivalent veltuzumab and rituximab fail to alter the levels ofphosphorylated p38 and PTEN from untreated control, whereas all 3 HexAbsincrease phosphorylated p38 and PTEN levels significantly. Similarresults were obtained in Raji cells for the decrease in phosphorylatedERKs and the increase in phosphorylated p38. No appreciable change inthe basal expression of signaling molecules was observed in Daudi cellsupon ligation of CD22 by epratuzumab. (3) The apoptosis and inhibitionof cell proliferation resulting from crosslinking veltuzumab orrituximab with GAH involves signaling events that are distinguishablefrom those associated with the HexAbs, as manifested in phosphorylatedERK (increase vs decrease), intracellular calcium (increase vs nochange), and mitochondrial membrane potential (loss vs no change).

For example, we showed that all 3 HexAbs at 10 nM and the bivalentveltuzumab or rituximab at 133 nM, but not at 10 nM, produced similarresults in the observed levels of various phosphorylated proteins, CDKinhibitors, and Bcl-2 family members that are known to mediateproliferation, cell-cycle arrest, and apoptosis. Specifically, weobserved a notable decrease in p-Lyn, p-Akt, p-BAD, p-ERK1/2, p-IKKα/β,p-IKBα, Mcl-1, Bcl-2, and Bcl-x1 levels in the treated versus untreatedcells, indicating that multiple prosurvival pathways were negativelyaffected, which require a higher threshold for the bivalent antibodies.On the other hand, we also noted that a significant increase in thepro-apoptotic signals (phosphorylated p38 and Bax), the tumor suppressor(PTEN), and the CDK inhibitors (p21 and Kip2) was only observed with theHexAbs. Moreover, only the HexAbs induced G₁ arrest, which apparentlyaugmented their antiproliferative potency. However, clustering of CD20or both CD20 and CD22 via the HexAbs induced neither a rapid rise inintracellular calcium nor phosphorylation of Lyn, Syk, and PLCγ, whichare characteristic of ligating BCR with anti-IgM (Niiro & Clark, Nat RevImmunol 2002, 2:945-956). The inability of the HexAbs to effect atransient increase in intracellular calcium as well as a notableΔψA_(m), and the down-regulation rather than up-regulation ofphosphorylated ERK, also differentiate their action from that induced bycrosslinking veltuzumab or rituximab with a secondary antibody.

Additional studies using PTEN siRNA and the PI3K inhibitor, LY294002,suggest that PTEN, which converts PI(3,4,5)P₃ to PI(4,5)P₂, and PI3K,producing PI(3,4,5)P₃ from PI(4,5)P₂, play opposing roles in mediatingthe direct toxicity induced by the anti-CD20/CD22 HexAbs, for which weshow PTEN siRNA mitigates, whereas LY294002 enhances, the apoptoticoutcome. Such results suggest that accumulation of PI(4,5)P₂, either viathe up-regulation of PTEN or the inhibition of PI3K, may be critical fortipping the balance of survival toward death.

Collectively, our findings are consistent with the view that the potentdirect cytotoxicity of 20-20, 22-20, and 20-22 is due to their abilityfor multivalent binding, which lowers the threshold for modifyingmultiple signaling pathways, resulting in a new distribution of pro- andanti-apoptotic proteins that promotes growth arrest, apoptosis, and,eventually, cell death. Surprisingly, these effects translated tonotable differences with regard to their relative potency for killingnormal human B cells versus human Burkitt lymphoma cells ex vivo, where22-20 and 20-22 showed a higher therapeutic ratio (percentage killing ofmalignant vs percentage normal B cells), compared with veltuzumab andrituximab (Rossi et al., Blood 2009, 113:6161-6171).

Because neither 22-20 nor 20-22 displayed CDC activity, in contrast tothe parental veltuzumab, and although 22-20 was less effective in ADCCthan 20-22 or veltuzumab, the presence of 4 Fabs of veltuzumab in 22-20enhanced the ADCC of epratuzumab, which was found to be moderate butstatistically significant (Rossi et al., Blood 2009, 113:6161-6171).Taking into consideration that both 22-20 and 20-22 had a highertherapeutic index ex vivo in terms of relative killing of lymphomaversus normal B cells than their parental antibodies, we speculate thata bispecific anti-CD20/CD22 HexAb may be a more potent class ofantilymphoma therapeutic antibodies for clinical use. In terms oftreating B-cell lymphomas and leukemias expressing both CD20 and CD22,the results suggest that 22-20, with 4 anti-CD20 Fabs, would be the moreeffective of the 2 bsAb choices. These experiences stimulate us to studywhether bispecific HexAbs against other cancer targets also can acquiredifferent and improved therapeutic properties over their parentalbivalent antibody forms.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention.

1. A method of killing CD20⁺ and/or CD22⁺ cells, comprising exposing thecells to a hexavalent DNL complex comprising: a. a first fusion proteincomprising an AD moiety from the N-terminus of an AKAP proteinconsisting of SEQ ID NO: 16 attached to the C-terminal end of an IgGantibody, and b. four copies of a second fusion protein comprising a DDDsequence from human protein kinase A (PKA) RIα, RIβ, RIIα or RIIβconsisting of SEQ ID NO: 14 attached to the C-terminal end of a Fabantibody; wherein pairs of the DDD sequence form dimers that bind to theAD moiety to form the DNL complex; and wherein the IgG antibody and theFab antibody fragment bind to one or more antigens selected from thegroup consisting of CD20 and CD22.
 2. The method of claim 1, wherein theIgG antibody is an anti-CD20 or an anti-CD22 antibody.
 3. The method ofclaim 2, wherein the Fab antibody fragment is a Fab fragment of ananti-CD20 or an anti-CD22 antibody.
 4. The method of claim 1, whereinthe anti-CD20 antibody is hA20 and the anti-CD22 antibody is hLL2. 5.The method of claim 1, wherein the anti-CD20 or anti-CD22 antibody ornaked Fab fragment thereof is a naked antibody or fragment thereof. 6.The method of claim 5, further comprising exposing the cell to at leastone therapeutic agent selected from the group consisting of a cytotoxin,a chemotherapeutic agent, a drug, a pro-drug, a toxin, an enzyme, animmunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, acytokine, and a hormone.
 7. The method of claim 1, wherein the anti-CD20or anti-CD22 antibody or Fab fragment thereof is further conjugated toat least one therapeutic agent selected from the group consisting of aradionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a pro-drug,a toxin, an enzyme, an immunomodulator, an anti-angiogenic agent, apro-apoptotic agent, a cytokine, and a hormone.
 8. The method of claim6, wherein the therapeutic agent is selected from the group consistingof aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,cisplatinum, irinotecan (CPT-11), SN-38, carboplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vinblastine, vinorelbine, vincristine, ricin,abrin, ribonuclease, ranpirnase, rapLR1, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.
 9. The method of claim6, wherein the therapeutic agent is bortezomib.
 10. The method of claim7, wherein the therapeutic agent is a radionuclide selected from thegroup consisting of ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt,¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ¹¹C, ^(121m)Te, ^(122m)Te, ¹²⁵I,^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹³N, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm,¹⁵O, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au,¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po,²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²²⁵Fm, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr,⁵⁷Cr, ⁵⁷Co, 58Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br,^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo and ^(99m)Tc.
 11. The method ofclaim 6, wherein the therapeutic agent is an enzyme selected from thegroup consisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.
 12. The method of claim 6, wherein the therapeuticagent is an immunomodulator selected from the group consisting oferythropoietin, thrombopoietin, tumor necrosis factor-α (TNF), TNF-β,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, stem cell growth factor designated “S1factor”, human growth hormone, N-methionyl human growth hormone, bovinegrowth hormone, parathyroid hormone, thyroxine, insulin, proinsulin,relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), luteinizing hormone (LH), hepatic growthfactor, prostaglandin, fibroblast growth factor, prolactin, placentallactogen, OB protein, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, LIF, FLT-3, angiostatin, thrombospondin, endostatin andlymphotoxin lymphotoxin (LT).